CN112996536A - H52IBV vaccine with heterologous spike protein - Google Patents

H52IBV vaccine with heterologous spike protein Download PDF

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CN112996536A
CN112996536A CN201980071601.2A CN201980071601A CN112996536A CN 112996536 A CN112996536 A CN 112996536A CN 201980071601 A CN201980071601 A CN 201980071601A CN 112996536 A CN112996536 A CN 112996536A
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A·克雷默-屈尔
E·S·蒙特
H-C·菲利普
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Boehringer Ingelheim Vetmedica GmbH
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Abstract

The present invention relates to H52IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or a fragment thereof. Further, the present invention relates to an immunogenic composition comprising said H52IBV encoding a heterologous S (spike) protein or a fragment thereof. Furthermore, the present invention relates to a method for immunizing a subject comprising administering to said subject an immunogenic composition of the invention. Furthermore, the present invention relates to a method of treating or preventing clinical signs caused by IBV in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to the invention.

Description

H52IBV vaccine with heterologous spike protein
Sequence listing
The present application contains a sequence listing according to 37 c.f.r.1.821-1.825. The sequence listing accompanying this application is incorporated herein by reference in its entirety.
Background
Avian coronavirus Infectious Bronchitis Virus (IBV) is the prototype gamma coronavirus of the family coronaviridae of the order nidovaviridae (Nidovirales). Infectious bronchitis viruses mainly infect the upper respiratory epithelium of chickens, causing respiratory diseases, often complicated by secondary bacterial pathogen infections (Cook et al 2012.Avian Pathol.41: 239-. Some IBV strains also affect the renal tubules, oviducts and parts of the gastrointestinal tract, leading to pathological lesions and clinical signs in these organ systems. The virus is widely present in commercial poultry and poultry in the courtyard. IBV is classified into a variety of genotypes, serotypes, and protective types due to its high genomic variability. IBV is currently considered to be one of the most economically relevant viral pathogens in the poultry industry.
The infectious bronchitis virus is an enveloped virus having a forward single-stranded RNA genome of 27.6kb (Cavanagh 2007.vet. Res.38: 281-. The first two thirds of the viral genome comprise a large coding region (also referred to as gene 1) divided into two open reading frames 1a and 1b, which encode 15 non-structural proteins involved in RNA replication, editing and transcription. The last third of the viral genome encodes the structural proteins: spike protein (S, encoded by gene 2), envelope protein (E, encoded by gene 3 c), membrane protein (M, encoded by gene 4), nucleocapsid protein (N, encoded by gene 6). Proteins S, E and M are part of the viral envelope, while protein N forms the ribonucleoprotein core with the viral RNA. The coronavirus spike protein determines the tropism of the host species (Kuo et al 2000.J. Virol.74: 1393-. It is a dimeric or trimeric transmembrane protein, which is proteolytically cleaved into two subunits, S1 and S2. The highly glycosylated S1 domain forms the "head" of the spike protein, containing a receptor binding domain that interacts with 2, 3-linked sialic acid on the surface of the host cell (Promkend et al 2014.virology.448: 26-32). The S2 domain includes the remainder of the ectodomain (ectomain) ("stem"), the transmembrane domain, and an endodomain located in the cytoplasm.
To date, the most widely used live attenuated IBV vaccine strains were developed in the 1960 s in the Netherlands by serial passages of Massachusetts-like IBV strains (Bijlenga et al 2004; Avian Pathol. 33: 550-. However, since the 1970 s, new IBV serotypes emerged that were not adequately protected by the traditional Massachusetts-like vaccines (Cook et al 2012.Avian Pathol.41: 239-) -250). Thus, there remains a need for new and highly effective IBV vaccines against other IBV serotypes.
IBV Beaudette (Geilhausen et al 1973.Arch Gesamte Virusforssch.: 40(3) (1973)), pp. 285-. However, attenuation may result in loss of immunogenicity.
In addition, recombinant IBV has been generated. Zhou et al 2016(Arch virol.; 161: 3179-. Hodgson et al 2004(J Virol 78: 13804-13811) discloses Beaudette (Massachusetts genotype) IBV with a spike protein of M41(Massachusetts genotype). Furthermore, Armesto et al.2011(PLoS One:6(8): e24352) discloses IBV Beaudette (Massachusetts genotype) with a heterologous spike protein from 4/91(4/91 genotype).
However, neither the recombinant IBVs disclosed in Zhou et al 2016 nor Hodgson et al 2004 could be considered IBVs with heterologous spike proteins, since both the IBV and the inserted spike protein are from the same genotype/serotype (Massachusetts). Again, all mentioned vaccines are based on the scaffold of Beaudette or have spike proteins from Beaudette.
Furthermore, although Beaudette has been described decades ago and recombinant methods known to use Beaudette have been described for more than decades, no Beaudette-based vaccine and no such recombinant vaccine (with heterologous spike proteins) are commercially available. Recombinant IBV based on Beaudette is not suitable as a vaccine. Wei et al 2014(Apl Microbiol Biotechnol 98) discloses Beaudette IBV with the S1 subunit of H120.
Ellis et al 2018(J.Virol.92(23)), Hodgson et al (J.Virol.78(24)), and Armesto et al 2011(PLoS One:6(8): e24352) all disclose Beaudette IBVs having M41 or 4/91 spike proteins. However, Ellis et al 2018(J.Virol.92(23)) described a recombinant Beaudette with a heterologous S1 subunit from M41 or QX combined with a chimeric spike protein of the Beaudette spike protein S2 subunit, which did not provide sufficient protection against S1 homology challenge ("a single vaccination of specific pathogen-free chickens with an rIBV expressing the virulent strains M41 or QX, S1, i.e. BeauR-M41(S1) and BeauR-QX (S1), providing incomplete protection against homologous challenge based on ciliary activity and clinical signs"; abstract "). Further, Ellis et al 2018(j.virol.92(23)) described that the full-length S gene (S1 and S2 from M41) only provides partial protection against challenge with homologous serotype IBV (page 12), suggesting that the IBV Beaudette strain is not suitable as a scaffold for recombinant IBV vaccines. Hodgson et al (j. virol.78(24)) further discloses that the Baudette strain is "also considered less immunogenic", and thus "it has never been used as a vaccine strain" (page 13802, left column, second paragraph). Therefore, there is a need to produce new high-potency IBV vaccines and recombinant IBV vaccines, respectively. Furthermore, there is a need for highly effective IBV vaccine vectors.
Detailed Description
Before describing aspects of the present invention, it must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a plurality of antigens, reference to "a virus" refers to one or more viruses and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing cell lines, vectors and methodologies that are reported in the publications that might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Composition of matter
The present invention solves the problems inherent in the prior art and provides a significant advance in the art.
In general, the invention provides H52IBV (infectious bronchitis virus) encoding a heterologous IBV S (spike) protein or fragment thereof.
The term "H52 IBV" is well known to those skilled in the art. The term "IBV" refers to infectious bronchitis virus. The term "H52" defines a particular strain of IBV. The H52 strain is well known to those skilled in the art and belongs to the Massachusetts genotype. IBV strains are usually distinguished by the coding sequence of the S1 subunit of the spike protein (Valastro et al.2016. infection Genet Evol.39:349-364), but can also be distinguished by their complete nucleotide sequence or by the sequence of specific proteins such as spike proteins, nucleocapsid proteins, envelope (E) proteins or membrane (M) glycoproteins. Since the spike protein determines the host tropism and antigenicity of IBV, the IBV genotype is classified by the coding sequence of spike protein subunit 1. Alternatively, IBV strains may be distinguished by their serotype. Serotype classification involves treatment of the virus with neutralizing antibodies.
Furthermore, H52 and H120 can be distinguished by higher pathogenicity after application to young chickens.
How to obtain H52IBV is common knowledge of the skilled person. H52IBV strains are commercially available, such as exemplified Nobilis IB H52(MSD Animal Health), AviPro IB 52(Lohmann Animal Health GmbH & co.kg), bronhovac (ceva), and the like. Furthermore, McDonald et al 1980(Avain Pathology 9: 245-. Furthermore, H52IBV has been used as a vaccine strain for decades (Bijlenga et al 2004, Avian Pathology 33(6): 550-. Methods for isolating the H52IBV strain and identifying the H52IBV strain are well known to those skilled in the art. For example, H52IBV strains can be identified as described in Zwaagstra et al 1992(J.Clin. Microbiol.30(1):79-84), Handberg et al 1999(Avian Pathology 28: 327) -335) or Callison et al 2006(Journal of viral Methods 138: 60-65). Zwaagstra et al 1992 and Handberg et al 1999 for example disclose Massachusetts specific primers (directed against S and N proteins, respectively) for RT-PCR and sequencing and reference sequences for comparison. In addition, H52IBV has been sequenced and genomic sequences, such as EU817497, can be obtained. Thus, the viral genome can be produced by synthesizing its sequence, and can be produced by applying a reverse genetics system.
The term "spike protein" refers to a specific protein that is IBV well known to those skilled in the art. Spike proteins are the main inducers of antibodies and protective immune responses. In addition, the spike (S) protein facilitates entry of IBV into the cell by binding to the cellular receptor of the host cell and also by mediating virus-cell membrane fusion with the host cell. Furthermore, it determines the tissue and cellular tropism of the virus strain.
The term "heterologous S (spike protein)" refers to a spike protein or fragment thereof that has been introduced into H52IBV from a different genotype or serotype than H52 IBV. Because H52 is a Massachusetts genotype and serotype, the heterologous spike protein is a non-Massachusetts genotype or serotype.
The terms "protein", "amino acid" and "polypeptide" are used interchangeably. The term "protein" refers to an amino acid sequence consisting of naturally occurring amino acids and derivatives thereof. Naturally occurring amino acids are well known in the art and are described in standard textbooks of biochemistry. Within the amino acid sequence, the amino acids are linked by peptide bonds. In addition, the two ends of the amino acid sequence are referred to as the carboxyl end (C-terminal) and the amino end (N-terminal). The term "protein" includes substantially purified proteins or protein preparations that additionally comprise other proteins. Furthermore, the term also relates to protein fragments. Furthermore, it includes chemically modified proteins. Such modifications may be artificial or naturally occurring modifications, such as phosphorylation, glycosylation, myristoylation, and the like.
Further, the present invention also provides an immunogenic composition comprising H52IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or a fragment thereof.
Furthermore, the present invention provides immunogenic compositions comprising IBV (infectious bronchitis virus) as described herein. Further, the present invention also provides an immunogenic composition comprising H52IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or a fragment thereof.
The term "immunogenic composition" refers to a composition comprising at least one antigen that elicits an immune response in a host to which the immunogenic composition is administered. Such an immune response may be a cellular and/or antibody-mediated immune response to the immunogenic composition of the invention. Preferably, the immunogenic composition induces an immune response, more preferably confers protective immunity against one or more clinical signs of IBV infection. The host is also referred to as an "object". Preferably, any host or subject described or referred to herein is avian or poultry.
Generally, an "immune response" includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells and/or cytotoxic T cells and/or γ - δ T cells specific for one or more antigens comprised in the immunogenic composition of the invention. Preferably, the host will exhibit a protective immune response or therapeutic response.
A "protective immune response" or "protective immunity" is evidenced by a reduction or disappearance of clinical signs typically exhibited by an infected host, a faster recovery time and/or a shortened duration of infection by an infected host, or a reduction in pathogen titer in tissues or body fluids or excretions.
An immunogenic composition is described as a "vaccine" if it exhibits a protective immune response in the host, whereby resistance to new infections is enhanced and/or the clinical severity of the disease is reduced.
H52-IBV-is defined by the protein coding sequence
In another particular aspect of the IBV or immunogenic composition of the invention, the H52IBV has a nucleotide sequence as shown in EU817497(SEQ ID NO:78) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
The term "nucleic acid" or "nucleic acid sequence" or "nucleotide sequence" refers to a polynucleotide comprising a DNA molecule, an RNA molecule, a cDNA molecule or a derivative. The term encompasses single-stranded and double-stranded polynucleotides. Nucleic acids of the invention include isolated polynucleotides (i.e., isolated from their natural environment) and genetically modified forms. Also included are chemically modified polynucleotides, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides, such as biotinylated polynucleotides. Furthermore, the terms "nucleic acid" and "polynucleotide" are used interchangeably to refer to any nucleic acid. The terms "nucleic acid" and "polynucleotide" also specifically include nucleic acids consisting of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).
The term "RNA" refers to any ribonucleic acid. The term encompasses single-stranded and double-stranded RNA. The RNA of the present invention includes both isolated RNA (i.e., isolated from its natural environment) and genetically modified forms. In addition, chemically modified RNA is also included, including naturally occurring modified RNA, such as methylated RNA, or artificially modified RNA, such as biotinylated RNA. The term "RNA" also specifically includes RNAs consisting of bases other than four biologically occurring nucleotides/bases (adenine, guanine, cytosine and uracil).
In another particular aspect of the IBV or immunogenic composition of the invention, the H52IBV strain has a spike (S) protein having an amino acid sequence as shown in AF352315(SEQ ID NO:79) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
It must be understood that the spike protein or nucleic acid sequence may be used to determine whether any IBV strain is derived from H52. However, since H52IBV is used as the backbone and the H52 spike protein or nucleic acid sequence is replaced by a heterologous spike protein or fragment thereof, the final IBV with the heterologous spike protein does not comprise any H52 spike protein or only a remaining portion of the H52 spike protein.
In another particular aspect of the IBV or immunogenic composition of the invention, the H52IBV strain has a spike (S) protein having an amino acid sequence as set forth in SEQ ID No. 1 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
In another particular aspect of the IBV or immunogenic composition of the invention, the H52IBV strain has a nucleocapsid (N) protein having an amino acid sequence as shown in AY044185(SEQ ID NO:80) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to at least one of the above sequences.
In another particular aspect of the IBV or immunogenic composition of the invention, the H52IBV strain has a nucleocapsid (N) protein having an amino acid sequence as shown in AF352310(SEQ ID NO:81) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
In another particular aspect of the IBV or immunogenic composition of the invention, the H52IBV strain has a nucleocapsid (N) protein having an amino acid sequence as shown in SEQ ID No.2 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
In another particular aspect of the IBV or immunogenic composition of the invention, the H52IBV strain has an envelope (E) protein having an amino acid sequence as shown in AF317210(SEQ ID NO:82) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
In another particular aspect of the IBV or immunogenic composition of the invention, the H52IBV strain has an envelope (E) protein having an amino acid sequence as shown in SEQ ID No. 3 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
In another particular aspect of the IBV or immunogenic composition of the invention, the H52IBV strain has a membrane glycoprotein (M) having an amino acid sequence as shown in AF286185(SEQ ID NO:83) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
In another particular aspect of the IBV or immunogenic composition of the invention, the H52IBV strain has a membrane glycoprotein (M) having an amino acid sequence as shown in SEQ ID No. 4 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
The term "identity" or "sequence identity" is known in the art and refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, i.e., the relationship between a reference sequence and a designated sequence to be compared to the reference sequence. After optimal alignment of a given sequence with a reference sequence to produce the highest degree of sequence similarity, sequence identity is determined by sequence comparison, as determined by the match between strings of sequence characters. Based on this alignment, sequence identity can be determined on a position-by-position basis, e.g., if at a position a nucleotide or amino acid residue is identical, then at that particular position the sequence is "identical". The total number of such positional identities is divided by the total number of nucleotides or residues in the reference sequence to yield the percentage of sequence identity. Sequence identity can be readily calculated by known methods including, but not limited to, those described in comparative Molecular Biology, Lesk, A.N., ed., Oxford University Press, New York (1988), Biocomputing: information and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey (1994); sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); sequence Analysis Primer, Gribskov, m. and Devereux, j., eds., m.stockton Press, New York (1991); and those described in Carillo, h., and Lipman, d., sia j. applied math, 48:1073(1988), the teachings of which are incorporated herein by reference. Preferred methods of determining sequence identity are designed to give the greatest match between the sequences tested. Methods for determining sequence identity are compiled in publicly available computer programs for determining sequence identity between designated sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research,12(1):387(1984)), BLASTP, BLASTN, and FASTA (Altschul, S.F.et al., J.Molec. biol.,215: 403-. The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S.et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S.F.et al., J.Molec. biol.,215: 403-. These programs use default gap weights to optimally align sequences to yield the highest level of sequence identity between the designated sequence and the reference sequence. For example, a polynucleotide having a nucleotide sequence that has at least, e.g., 85%, preferably 90%, even more preferably 95% "sequence identity" to a reference nucleotide sequence means that the nucleotide sequence of the specified polynucleotide is identical to the reference sequence, except that the specified polynucleotide sequence may include at most 15, preferably at most 10, even more preferably at most 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence that is at least 85%, preferably 90%, even more preferably 95% identical to a reference nucleotide sequence, at most 15%, preferably 10%, even more preferably 5% of the nucleotides in the reference nucleotide sequence may be deleted or substituted with another nucleotide, or at most 15%, preferably 10%, even more preferably 5% of the nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed between individual nucleotides in the reference sequence, or in one or more contiguous groups within the reference sequence. Similarly, a polypeptide having an amino acid sequence that has at least, e.g., 85%, preferably 90%, even more preferably 95% sequence identity to a reference amino acid sequence, means that the amino acid sequence of the specified polypeptide is identical to the reference sequence, except that the specified polypeptide sequence can include at most 15, preferably at most 10, even more preferably at most 5 amino acid changes per 100 amino acids of the reference amino acid sequence. In other words, in order to obtain a given polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity to a reference amino acid sequence, at most 15%, preferably at most 10%, even more preferably at most 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or at most 15%, preferably at most 10%, even more preferably at most 5% of the total number of amino acids in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence can occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed among the individual residues in the reference sequence, or in one or more contiguous groups within the reference sequence. Preferably, conservative amino acid substitutions at residue positions that are not identical are different. However, conservative substitutions are not included as a match in determining sequence identity.
The terms "identity", "sequence identity" and "percent identity" are used interchangeably herein. For the purposes of the present invention, it is defined herein that in order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid or nucleotide residues at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,% identity is the number of identical positions/total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are of the same length.
Sequence comparisons can be made over the full length of the two sequences being compared or over fragments of the two sequences. Typically, the comparison is made over the full length of the two sequences being compared. However, sequence identity may be compared over a region of, for example, 20, 50, 100 or more contiguous amino acid residues.
The skilled person will appreciate that different computer programs can be used to determine homology between two sequences. For example, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the following algorithm: needleman and Wunsch (J.mol. biol. (48):444-453(1970)) algorithm, which has been incorporated into the GAP program in the Accelrys GCG software package (available from http:// www.accelrys.com/products/GCG /), using Blosum 62 matrix or PAM250 matrix, GAP weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2,3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results when using different algorithms, but that the overall percent identity of two sequences will not change significantly.
The protein sequences or nucleic acid sequences of the invention may further be used as "query sequences" to search public databases, for example to identify other family members or related sequences. Such a search can be performed using BLASTN and BLASTP programs (version 2.0) as described in Altschul, et al (1990) J.mol.biol.215: 403-10. A BLAST protein search can be performed using the BLASTP program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain Gapped alignments for comparison purposes, Gapped BLAST programs can be used, as described in Altschul et al (1997) Nucleic Acids Res.25(17): 3389-3402. When BLAST and Gapped BLAST programs are used, the default parameters for each program (e.g., BLASTP and BLASTN) can be used. See National Center for Biotechnology Information homepage http:// www.ncbi.nlm.nih.gov/.
As used herein, the term "identical to the sequence of SEQ ID NO: X" is to be understood in particular as being equivalent to the term "identical to the sequence of SEQ ID NO: X in length" or "identical to the sequence of SEQ ID NO: X in full length". As used herein, "X" is any integer selected from 1-85, whereby "SEQ ID NO: X" means any of the SEQ ID NO's mentioned herein.
Heterologous S protein
IBV strains
IBV strains can be classified by serotype and genotype. Serotype classification involves treatment of the virus with neutralizing antibodies, while genotype classification typically involves testing the sequence of the S1 (spike) protein. However, different strains of IBV are well known to those skilled in the art. Infectious bronchitis virus was first discovered in the united states in the 1930 s. The first IBV serotype identified was Massachusetts type, but several other serotypes were identified in the united states, including Arkansas and Delaware, in addition to the originally identified Massachusetts type.
The IBV strain Beaudette is of the Massachusetts type and is produced by passage through at least 150 passages in chicken embryos. The IBV strain Beaudette was originally isolated from Beaudette and Hudson (J.am.vet.Med.A.90,51-60,1937) and passaged in chicken embryos. Other Massachusetts type IBV strains than Beaudette are H120, H52 and M41. The H120 strain was passaged for 120 passages.
IBV QX is described as a virulent local isolate of IBV, originally isolated in china. However, the virus has spread to europe and has been found in parts of western europe, mainly in the netherlands, but also in germany, france, belgium, denmark and the uk. In addition, QX genotypes or serotypes have been described in some countries of asia and africa.
IBV 4/91, commonly known as 793B, was first reported in the uk in the early 90 s and has now spread to many parts of the world. CR88 is an attenuated strain belonging to this serotype and is commercially available as a vaccine.
In late 1990 s, strains designated "Italian-02" or "Italy-02" were isolated in Italy. Sequence analysis of one of these isolates was published in 2002 (NCBI-BLAST, accession number AJ 457137). However, studies have shown that this Italian-02 strain is very common in Europe and has become one of the most prominent genotypes in the United kingdom, Spain, France and the Netherlands, with the exception of the IBV variant 4/91.
Since 1996, a new Infectious Bronchitis Virus (IBV) genotype, designated Q1, was prevalent in china and first reported in italy in 2011. Q1 was associated with increased mortality, renal pathology and proventriculitis.
Furthermore, strains D274, B1648/D8880, D1466, V1397 and Arkansas were also identified in Europe.
How to obtain any strain of IBV is common knowledge to the skilled person. IBV strains are commercially available, obtained from scientific research institutions, and can be used because the IBV strain has been sequenced and the sequence has been published, and the genome can also be synthesized as complementary DNA. This is achieved byIn addition, IBV strains can be isolated from the field. Methods for isolating and identifying IBV strains are well known to those skilled in the art. Valter Leonardo de Quadros 2011 (Disservation, Das)
Figure BDA0003043693370000091
Bronchitis Virus(IBV):Molekularbiologische Untersuchungen zur Diagnostik und zum Vorkommen sowie zur
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des Genotyps IBV QX in spezifisch pathogenfreien (SPF)Broilern,Freie
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Berlin), Worthington et al 2009(Avian Pathology 37(3), 247-.
In another particular aspect of the IBV or immunogenic composition of the invention, the heterologous spike protein is a non-Massachusetts genotype or serotype.
In another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from an IBV having a genotype or serotype selected from the group consisting of: arkansas (e.g., Arkansas 99), Brazil (e.g., BR-1, BR-2, 23/2013, IBV/Brasil/351/1984), California (e.g., California1734/04, California99), Connecticut, Delaware (e.g., Delaware 98), Dutch (e.g., D207, D212, D274, D3128, D3896, D8880, D1466), Florida, Georgia (e.g., Georgia GA-07, GA-08, GA-12, GA-13), Gray, Holte, Iowa (e.g., Iowa 97 and Iowa 69), Italy (e.g., Italy 02), JMK, IBT 3, Maine (e.g., Mannsylvanine 209), Pennsylvania (e.g., Pennsylvania 1220/98, Pennsylvania/38998), Wolk (e.g., Wolk 084, Wolk 845), Wolk (e.g., CmvlSE/842, such as variants of Cqqqqqv/387), gamma CoV/Ck/Poland/G052/2016) and 4/91(793B, CR 88).
According to another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from an IBV selected from the group consisting of genotypes or serotypes: 4/91, QX, Q1, Italy 02, Arkansas, Connetictu, Georgia, LDT3, PL84084, variant 2 and Brazil.
According to another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from an IBV selected from the group consisting of genotypes or serotypes: 4/91, QX, Q1, Arkansas, variant 2 and Brazil.
According to another particular aspect of the IBV or immunogenic composition of the invention, the 4/91 strain is selected from the group consisting of: spain/98/328, Spain/92/35, IR-3654-VM, FR-CR88061-88, FR-85131-85, UK-1233-95, UK/3/91, Spain/00/336, UK/7/91, 4/91-pathogenic, 4/91 attenuated, IB4-91 and CR 88.
According to another particular aspect of the IBV or immunogenic composition of the invention, the QX strain is selected from the group consisting of: FR-L1450T-05, FR-L1450L-05, NL-L1449T-04, NL-L1449K-04, IBV/Ck/SP/170/09, IBV/Ck/SP/79/08, IBV/Ck/SP/248/09, HBN, IBVQX, LX4, BJQ, CK/CH/LGD/03 and GB 341/96.
According to another particular aspect of the IBV or immunogenic composition of the invention, the Q1 strain is selected from the group consisting of: CK/CH/LDL/98I, CK/CH/LSD/08-10, J2, Q1, AR08ER22, AR08BA21 and Chile-295-10.
According to another particular aspect of the IBV or immunogenic composition of the invention, the Arkansas strain is selected from the group consisting of: ark99, ArkGA, ArkDPI, AL/5364/00, ARKDPI11, AL/0803/01, AL/7149/00, ArkDPI101, AL/1221/01, AL/1793/01 and AL/4614/98.
According to another particular aspect of the IBV or immunogenic composition of the invention, the variant 2 strain is selected from the group consisting of: IS/1494/06, IBV/Ck/EG/CU/4/2014, gamma CoV/Ck/Poland/G052/2016, Eg/CLEVB-2/IBV/012, D1344/2/4/10_ EG, TR8 and IB VAR 2-06.
According to another particular aspect of the IBV or immunogenic composition of the invention, the Brazil strain is selected from the group consisting of: BR-1, BR-2, 23/2013 and IBV/Brasil/351/1984.
According to another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from 4/91 genotype or serotype.
In another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from the 4/91 genotype or serotype, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID No. 5 or 6.
In another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from a QX genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID No. 7 or 8.
In another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from the Q1 genotype or serotype, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID No. 9 or 10.
In another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from the Arkansas genotype or serotype, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity with SEQ ID No. 11 or 12.
In another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from a variant 2 genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID No. 13 or 14.
In another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is from a Brazil genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID No. 15 or 16.
In another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein or fragment thereof is selected from SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.
In another particular aspect of the IBV or immunogenic composition of the invention, the heterologous S protein is a full length spike protein.
The experimental data of the present invention indicate that fragments of the spike protein sequence, such as the extracellular domain of the spike protein, can be used. However, full-length spike protein sequences may also be used.
In another particular aspect of the IBV or immunogenic composition of the invention, the fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1077 amino acids in length.
In another particular aspect of the IBV or immunogenic composition of the invention, the fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1077 amino acids in length from the N-terminus.
The term "N-terminal" is well known to those skilled in the art. The N-terminus is also referred to as the amino terminus, NH 2-terminus, N-terminus, or amine terminus. When a protein is translated from messenger RNA, it is produced from the N-terminus to the C-terminus. Thus, the N-terminus is the origin of the amino acid chain (protein or polypeptide) comprising the amine group (-NH 2).
In another particular aspect of the IBV or immunogenic composition of the invention, the fragment of the heterologous S (spike) protein is at least 1000 amino acids in length.
In another particular aspect of the IBV or immunogenic composition of the invention, the fragment of the heterologous S (spike) protein is the extracellular domain of the spike protein.
The term "extracellular domain" is well known to those skilled in the art. The spike protein comprises different functional parts, namely a signal sequence, an extracellular domain, a transmembrane domain and an intracellular domain (from N-terminus to C-terminus). Thus, after cleavage of the signal sequence, the N-terminus of the spike protein begins in the extracellular domain. The extracellular domain of the IBV spike protein is approximately 1077 amino acids in length, differing by several amino acids in length depending on the IBV strain.
In another particular aspect of the IBV or immunogenic composition of the invention, wherein the heterologous S (spike) protein or fragment thereof replaces the homologous S protein or fragment thereof.
In another particular aspect of the IBV or immunogenic composition of the invention, wherein the heterologous S (spike) protein or fragment thereof replaces a naturally occurring S protein or fragment thereof.
In another particular aspect of the IBV or immunogenic composition of the invention, wherein the heterologous S (spike) protein or fragment thereof replaces the S protein or fragment thereof in H52 IBV.
In another particular aspect of the IBV or immunogenic composition of the invention, the IBV is attenuated.
The term "attenuated" refers to a pathogen having reduced virulence compared to the wild-type isolate. In the present invention, an attenuated IBV is a strain in which toxicity has been reduced so as not to cause clinical signs of IBV infection but which is capable of inducing an immune response in a target animal, but may also refer to a reduction in the occurrence or severity of clinical signs in an attenuated IBV infected animal compared to a "control" animal that is not infected with an attenuated IBV or that does not receive an attenuated virus. Herein, the term "reduction" means a reduction of at least 10%, preferably 25%, even more preferably 50%, even more preferably 60%, even more preferably 70%, even more preferably 80%, even more preferably 90%, even more preferably 95%, most preferably 100% compared to a control group of non-attenuated IBV infection as defined above. Thus, an attenuated IBV strain is a strain suitable for incorporation into an immunogenic composition comprising a modified live IBV.
In another particular aspect of the IBV or immunogenic composition of the invention, the IBV is inactivated.
Any conventional inactivation method may be used for the purposes of the present invention. Thus, inactivation may be performed by chemical and/or physical treatment methods known to those skilled in the art. Preferred methods of inactivation include the addition of cyclized diethylene imine (BEI), including the addition of a solution of 2-bromoethyleneamine hydrobromide (BEA) that has been cyclized to diethylene imine (BEI). Preferred other chemical inactivators include, but are not limited to, Triton X-100, sodium deoxycholate, cetyltrimethylammonium bromide, beta-propiolactone, thimerosal, phenol, and formaldehyde (formalin). However, inactivation may also include a neutralization step. Preferred neutralizing agents include, but are not limited to, sodium thiosulfate, sodium bisulfite and the like.
Preferred formalin inactivation conditions include formalin concentrations of from about 0.02% (v/v) to about 2.0% (v/v), more preferably from about 0.1% (v/v) to about 1.0% (v/v), still more preferably from about 0.15% (v/v) to about 0.8% (v/v), even more preferably from about 0.16% (v/v) to about 0.6% (v/v), and most preferably from about 0.2% (v/v) to about 0.4% (v/v). The incubation time depends on the resistance to IBV. Typically, the inactivation procedure is performed until no IBV growth is detected in a suitable culture system.
Preferably, the inactivated IBV of the invention is formalin inactivated, preferably using the concentrations described herein above.
The inactivated IBV of the invention may be incorporated into liposomes using known techniques, for example as described in Nature 1974, 252-254 or Journal of Immunology,1978,120,1109-13. In another embodiment of the invention, the inactivated IBV of the invention may be conjugated with a suitable biological compound such as a polysaccharide, a peptide, a protein, etc. or a combination thereof.
In another particular aspect of the IBV or immunogenic composition of the invention, the IBV is genetically engineered.
The term "genetically engineered" refers to an IBV that has been mutated by using a "reverse genetics" approach. Preferably, the IBV of the invention has been genetically engineered. Reverse genetics techniques involve the preparation of synthetic recombinant viral RNA. However, "reverse genetics" techniques are well known to those skilled in the art.
In another particular aspect of the IBV or immunogenic composition of the invention, the IBV is a recombinant IBV.
As used herein, the term "recombinant" relates to an RNA genome (or RNA sequence, cDNA sequence or protein) having any modification that is not naturally occurring in the corresponding RNA genome (or RNA sequence, cDNA sequence or protein). For example, an RNA genome (or RNA sequence, cDNA sequence or protein) is considered "recombinant" if it contains insertions, deletions, inversions, relocations or point mutations that have been introduced artificially, e.g., by human intervention. Thus, an RNA genomic sequence (or an RNA sequence, a cDNA sequence or a protein) is not related to all or part of the sequence (or an RNA sequence, a cDNA sequence or a protein) with which it is naturally associated. The term "recombinant" as used with respect to a virus refers to a virus produced by the manual manipulation of the viral genome. The term "recombinant virus" includes genetically modified viruses.
In another particular aspect of the IBV or immunogenic composition of the invention, the IBV is chimeric.
The term "chimeric" refers to an IBV comprising one or more nucleotide sequences from another coronavirus, preferably from another IBV strain. For example, IBV H52 encoding a heterologous S (spike) protein or fragment thereof is a chimeric IBV.
In another particular aspect of the immunogenic composition according to the invention, the immunogenic composition is a vaccine. The term "vaccine" has been described elsewhere herein. However, if the host exhibits a protective immune response, whereby resistance to new infections is increased and/or the clinical severity of the disease is reduced, the immunogenic composition is described as a "vaccine".
In another particular aspect of the immunogenic composition according to the invention, the immunogenic composition comprises a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, adjuvants, immunostimulants, and combinations thereof.
"diluents" can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents may include sodium chloride, glucose, mannitol, sorbitol, lactose and the like. The stabilizer includes albumin, alkali metal salts of ethylene diamine tetraacetic acid, and the like.
In another particular aspect of the immunogenic composition of the invention, the pharmaceutically acceptable carrier is phosphate buffered saline.
Preferably, the immunogenic composition further comprises a sucrose gelatin stabilizer.
Preferably, the pharmaceutically acceptable carrier is chitosan.
Chitosan is a natural deacetylated polysaccharide of chitin derived from crustaceans (e.g., shrimp, crab), insects, and other invertebrates. Recently, Rauw et al in 2009 (Vet Immunol Immunop 134:249-258) demonstrated that chitosan enhances the cellular immune response of live vaccines against Newcastle disease, promoting its protective effect. Furthermore, Wang et al, 2012 (Arch Virol (2012)157: 1451-.
Preferably, the immunogenic composition may further comprise one or more other immune modulators, such as interleukins, interferons or other cytokines. The amounts and concentrations of adjuvants and additives useful within the scope of the present invention can be readily determined by the skilled artisan.
In certain aspects, the immunogenic compositions of the invention contain an adjuvant. As used herein, "adjuvants" may include aluminum hydroxide and aluminum phosphate, saponins such as Quil A, QS-21 (Cambridge Biotechnology, Massachusetts Cambridge), GPI-0100 (pharmaceutical preparations, Inc., Burming, Arbama), water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions. The emulsion may be based in particular on light liquid paraffin oil (typical of european medicines); isoprenoid oils such as squalane or squalene; oils produced from oligomers of alkenes, particularly isobutylene or decene. Esters of acids or alcohols containing straight chain alkyl groups, in particular vegetable oils, ethyl oleate, propylene glycol di (caprylate/caprate), glycerol tri (caprylate/caprate) or propylene glycol dioleate. Esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular sorbitol, mannitol (e.g. anhydromannitol oleate), ethylene glycol, polyglycerol, propylene glycol and esters of oleic, isostearic, ricinoleic or hydroxystearic acid, which may optionally be ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, in particular L121. See Hunter et al, theory and practice of adjuvants (Ed. Stutt-Ture, D.E.S.), John, Willd-son publishing Co., agricultural industry Standard, pp 51-94 (1995) and Tode et al, vaccine 15:564-570 (1997). Typical adjuvants are SPT emulsions as described in "vaccine design, subunit and adjuvant approach" page 147, editors are m.powell and m.newman, pleinan press, 1995, and MF59 emulsions as described in the same book, page 183.
Another example of an adjuvant is a compound selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and alkenyl derivatives. Advantageous adjuvant compounds are crosslinked polymers of acrylic or methacrylic acid, in particular crosslinked polymers with polyalkenyl ethers of saccharides or polyols. These compounds are called carbomers (carbomer) (volume 8, second, 6 months 1996). Those skilled in the art can also refer to U.S. Pat. No.2,909,462, which describes the crosslinking of such acrylic polymers with a polyol having at least 3 hydroxyl groups, preferably no more than 8 hydroxyl groups, the hydrogen atoms of at least 3 hydroxyl groups being substituted with an unsaturated aliphatic group having at least 2 carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl, and other ethylenically unsaturated groups. The unsaturated groups may themselves contain other substituents, such as methyl. The product sold under the name Carbopol (Carbopol) (BF Goodrich, ohio, usa) is particularly suitable. They are crosslinked with allyl sucrose or allyl pentaerythritol. Among these, mention may be made of carbopol 974P, 934P and 971P. Most commonly used is carbopol 971P. Among the copolymers of maleic anhydride and alkenyl derivatives, there is the copolymer EMA (monsanto company), which is a copolymer of maleic anhydride and ethylene. These polymers are dissolved in water to produce an acidic solution which will be neutralized, preferably to physiological pH, in order to prepare an adjuvant solution to be incorporated into an immunogenic, immunological or vaccine composition.
Further suitable adjuvants include, but are not limited to, RIBI adjuvant system (RIBI Corp.), Block copolymer (CytRx, Georgia, Atlanta), SAF-M (Chiron, Emmerrvier, Calif.), monophosphoryl lipid A, Avridine lipoamine adjuvant, heat labile endotoxin from E.coli (recombinant or other forms), cholera toxin, IMS 1314 or murine amido dipeptide, or naturally occurring or recombinant cytokines or analogs thereof, or stimulators of endogenous cytokine release, and the like.
It is contemplated that the adjuvant is added in an amount of about 100 micrograms to about 10 milligrams per dose, preferably about 100 micrograms to about 10 milligrams per dose, more preferably about 500 micrograms to about 5 milligrams per dose, even more preferably about 750 micrograms to about 2.5 milligrams per dose, and most preferably about 1 milligram per dose. In addition, the concentration of the adjuvant may be about 0.01 to 50%, preferably about 2 to 30%, more preferably about 5 to 25%, more preferably about 7 to 22%, and most preferably 10 to 20% by volume of the final product.
In another particular aspect of the immunogenic composition according to the invention, the immunogenic composition is effective in treating and/or preventing clinical signs caused by IBV in a subject in need thereof. The terms "treatment and/or prevention", "clinical symptoms" and "in need thereof have been defined elsewhere.
In another particular aspect of the immunogenic composition according to the invention, the immunogenic composition protects against challenge by an IBV strain of the genotype or serotype of the heterologous spike protein.
In another particular aspect of the immunogenic composition according to the invention, the immunogenic composition protects against challenge with 4/91, QX, Q1, Arkansas, variant 2 or Brazil genotype strains.
In another particular aspect of the immunogenic composition according to the invention, the immunogenic composition protects against challenge with the 4/91 genotype strain.
In another particular aspect of the immunogenic composition according to the invention, the immunogenic composition is formulated for single dose administration.
The volume of a single dose has been defined elsewhere herein.
Furthermore, it has been shown that one dose of the present immune composition is effective after administration of such a single dose of the present immune composition.
In another particular aspect of the immunogenic composition according to the invention, the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, by spraying, by drinking water or by eye drops.
In another particular aspect of the immunogenic composition according to the invention, the immunogenic composition comprises 1 to 10log per dose10 EID50The IBV of (1).
In another particular aspect of the immunogenic composition according to the invention, the immunogenic composition comprises 2 to 5log per dose10 EID50The IBV of (1).
In another particular aspect of the immunogenic composition according to the invention, the immunogenic composition comprises 2 to 4log per dose10 EID50The IBV of (1).
Reagent kit
If desired, the compositions may be presented in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient. For example, the package may comprise a metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration, preferably for administration to a subject, particularly poultry. Associated with such containers may be a notice written in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
Accordingly, the present invention provides a kit comprising an IBV or immunogenic composition as described herein.
In a particular aspect of the kit according to the invention, the kit further comprises instructions for the treatment and/or prevention of an avian disease.
In a particular aspect of the kit according to the invention, the kit further comprises instructions for the treatment and/or prevention of a disease in poultry.
In a particular aspect of the kit according to the invention, the kit further comprises instructions for the treatment and/or prevention of IB (infectious bronchitis).
Method of treatment
Furthermore, the present invention provides a method for immunizing a subject comprising administering to the subject an immunogenic composition as described herein.
The term "immunization" refers to active immunization by administering an immunogenic composition to a subject to be immunized, thereby eliciting an immune response against an antigen contained in such immunogenic composition.
Preferably, the immunization results in a reduction in the incidence of a particular IBV infection in the population or a reduction in the severity of clinical signs caused by or associated with a particular IBV infection.
Furthermore, immunization of a subject in need thereof with an immunogenic composition provided herein results in prevention of infection of the subject by IBV. Even more desirably, the result of immunization is an effective, long-lasting immune response to IBV infection. It will be appreciated that the period of time will last for more than 1 month, preferably more than 2 months, preferably more than 3 months, more preferably more than 4 months, more preferably more than 5 months, more preferably more than 6 months. It should be understood that immunization is not necessarily effective for all subjects being immunized. However, this term requires that a large proportion of subjects in a population be effectively immunized.
Preferably, a population of subjects is envisaged in this case, which will typically produce clinical signs, i.e. not immunized, typically caused by or associated with IBV infection. Whether a subject of the population is effectively immunized can be further determined by one of skill in the art. Preferably, the immunization should be effective if the incidence or severity of clinical signs in at least 33%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, more preferably at least 95%, most preferably 100% of the subjects of a population is reduced by at least 10%, more preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 100% compared to subjects not immunized or immunized with an immunogenic composition obtainable before the present invention but subsequently infected with the particular IBV. .
Furthermore, the present invention provides a method of treating or preventing clinical signs caused by IBV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition as described herein.
The term "treatment or prevention" refers to reducing the incidence of a particular IBV infection in a population, or reducing the severity of clinical signs caused by or associated with a particular IBV infection. Thus, the term "treating or preventing" also refers to a group of subjects receiving an effective amount of an immunogenic composition provided herein, reducing the number of subjects in the population infected with a particular IBV (i.e., reducing the incidence of infection with a particular IBV), or lessening the severity of clinical signs typically associated with or caused by infection with an IBV, or reducing viral shedding following infection with a particular IBV, or preventing or reducing egg drop in laying hens after infection with a particular IBV, as compared to a group of subjects not receiving such an immunogenic composition.
"treatment or prevention" generally refers to the administration of an effective amount of an immunogenic composition of the invention to a subject or population of subjects in need of or who may benefit from such treatment/prevention. The term "treatment" refers to the administration of an effective amount of an immunogenic composition once a subject or at least some subjects of the population have been infected with such IBV and wherein these subjects have shown some clinical signs caused by or associated with such IBV infection. The term "prevention" refers to administration to a subject prior to infection of the subject with IBV, or at least in the absence of any subject in the subject or group of subjects exhibiting any clinical signs caused by or associated with infection by IBV. The terms "preventing" and "prevention" are used interchangeably in this application.
As used herein, the term "effective amount" refers to, but is not limited to, an amount of antigen that elicits or is capable of eliciting an immune response in a subject. Such an effective amount can reduce the incidence of a particular IBV infection in a population or reduce the severity of clinical signs of a particular IBV infection.
Preferably, the incidence or severity of clinical symptoms is reduced by at least 10%, more preferably by at least 20%, more preferably by at least 30%, even more preferably by at least 40%, more preferably by at least 50%, even more preferably by at least 60%, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, more preferably by at least 95%, most preferably by 100% as compared to a subject not treated or treated with a previously useful immunogenic composition of the invention but subsequently infected with a particular IBV.
The term "clinical signs" as used herein refers to signs of IBV infection in a subject. The clinical signs of infection depend on the pathogen chosen. Examples of such clinical signs include, but are not limited to, respiratory distress, nephritis, salpingitis (salpingitis), egg abnormalities, feather upset, depression, decreased growth rate, and decreased appetite. Signs of respiratory distress include signs of respiration including asthma, cough, sneezing, tracheal rales, nasal and ocular secretions, tracheal lesions and ciliary retention (ciliostasis) in the trachea. Signs of nephritis include renal pathology and watery diarrhea. Signs of egg abnormalities include decreased egg size, smaller egg size, poorer eggshell, decreased internal quality of the egg, thinner egg albumen, and cilia stasis within the fallopian tubes. However, clinical signs also include, but are not limited to, clinical signs that can be observed directly from a living animal. Examples of clinical signs that can be directly observed from living animals include nasal and ocular secretions, coughing, wheezing, sneezing, tracheal rales, wrinkled feathers, conjunctivitis, weight loss, reduced growth rate, decreased appetite, dehydration, watery diarrhea, lameness, lethargy, emaciation, and loss of movement, among others.
Preferably, the reduction in the incidence or severity of clinical symptoms in a subject being treated refers to a reduction in cilia stasis, a reduction in rale, a reduction in egg drop, a reduction in renal pathology, a reduction in watery diarrhea, a reduction in weight loss, a reduction in viral load, a reduction in viral shedding, or a combination thereof, as compared to a subject not being treated or being treated with a previously useful immunogenic composition of the invention but subsequently infected with a particular IBV.
The term "in need of or" in need of "as used herein means that administration/treatment is associated with strengthening or improving health or clinical signs or any other positive medical effect on the health of a subject receiving the immunogenic composition of the invention.
The terms "reduce" or "reducing" or "mitigating or" reducing "may be used interchangeably in this application. The term "reduced" means that the clinical signs are reduced by at least 10%, more preferably by at least 20%, more preferably by at least 30%, even more preferably by at least 40%, more preferably by at least 50%, even more preferably by at least 60%, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, more preferably by at least 95%, most preferably by 100% compared to a subject not receiving treatment (not immunized) but subsequently infected with a particular IBV.
Furthermore, the present invention provides a method of reducing ciliary retention in a subject in need thereof, compared to a subject of an unimmunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition as described herein.
As shown in the examples, the immunogenic compositions provided herein have proven effective in reducing ciliary retention.
The term "cilia stasis" refers to a decrease in ciliary movement in the trachea. Thus, ciliary retention may be determined by examining the inner layer of the tracheal ring to determine ciliary movement. It is the general knowledge of the skilled person how to determine the movement of the cilia in the trachea.
Preferably, ciliary movement is not reduced from day 10 after challenge or infection, more preferably from day 5 after challenge or infection, more preferably from day 4 after challenge or infection, more preferably from day 3 after challenge or infection, and most preferably from day 1 or 2 after challenge or infection with IBV, as compared to subjects of an unimmunized control group of the same species.
The term "reduced cilia stasis" means that cilia stasis is reduced by at least 10%, preferably by at least 20%, more preferably by at least 30%, even more preferably by at least 40%, even more preferably by at least 50%, even more preferably by at least 60%, even more preferably by at least 70%, even more preferably by at least 80%, even more preferably by at least 90%, even more preferably by at least 95%, most preferably by 100% compared to subjects in an unvaccinated control group of the same species. It is the general knowledge of the skilled person how to measure the reduction of ciliary retention.
In one aspect of the invention, the subject is an avian species (avian).
The term "avian" is well known to those skilled in the art. The term "avian" includes all birds, including poultry.
In one aspect of the invention, the subject is poultry (poultry).
The term "poultry" is well known to those skilled in the art. The term "poultry" includes chickens (chicken), turkeys (turkey), quail (quail), pheasant (pheasant), guinea fowl (guineafowls), geese (goose) and ducks (duck). In addition, the term "chicken" includes broiler chickens, laying hens, and breeding hens of both, also known as breeder chickens.
In one aspect of the invention, the subject is selected from the group consisting of a chicken, turkey, quail or pheasant.
In one aspect of the invention, the subject is a chicken.
In one aspect of the invention, the immunogenic composition is administered once.
A single dose is understood to be administered only once. As shown in the examples, the immunogenic compositions provided herein have been demonstrated to be therapeutically effective after administration of a single dose to a subject in need thereof.
The dose volume per bird depends on the route of inoculation and the age of the bird.
Typically, eye drop vaccines are administered in volumes of 1 to 100 μ l per dose at any age. Preferably, the total volume of a single dose of the eye drop vaccine is between about 5. mu.l and 70. mu.l, more preferably between about 20. mu.l and 50. mu.l, preferably a single dose of 20. mu.l, 25. mu.l, 30. mu.l, 35. mu.l, 40. mu.l, 45. mu.l or 50. mu.l. Preferably, the total volume of a single dose of the eye drop vaccine is between about 5. mu.l and 70. mu.l, more preferably between about 20. mu.l and 50. mu.l, preferably a single dose of 20. mu.l, 25. mu.l, 30. mu.l, 35. mu.l, 40. mu.l, 45. mu.l or 50. mu.l.
For day-old poultry, the spray vaccine may comprise a dose volume of 25 to 1000 μ l. Preferably, the total volume of a single dose of the spray vaccine is between about 50. mu.l and 5000. mu.l, more preferably between about 75. mu.l and 2000. mu.l, more preferably between about 100. mu.l and 1000. mu.l, even more preferably between about 200. mu.l and 900. mu.l, more preferably between about 300. mu.l and 800. mu.l, even more preferably between about 400. mu.l and 700. mu.l, preferably a single dose of 400. mu.l, 425. mu.l, 450. mu.l, 475. mu.l, 500. mu.l, 525. mu.l, 550. l, 575. mu.l, 600. mu.l, 625. mu.l, 650. mu.l, 675. mu.l or 700. mu.l. Most preferably the total volume of a single dose is 400. mu.l, 450. mu.l, 500. mu.l, 550. mu.l, 600. mu.l, 650. mu.l or 700. mu.l.
Vaccines for in ovo vaccination may comprise a dose volume of 50 to 100 μ l, preferably 50 μ l. Preferably, the total volume of a single dose for an in ovo vaccine is from about 10 μ l to 250 μ l, more preferably from about 15 μ l to 200 μ l, even more preferably from about 20 μ l to 150 μ l, even more preferably about 30 μ l. 100 μ l, even more preferably between about 30 μ l and 75 μ l, and preferably a single dose of 30 μ l, 35 μ l, 40 μ l, 45 μ l, 50 μ l, 55 μ l, 60 μ l, 65 μ l, 70 μ l or 75 μ l. Most preferably, the total volume of a single dose is 40. mu.l, 45. mu.l, 50. mu.l, 55. mu.l or 60. mu.l.
A vaccine for intramuscular or subcutaneous vaccination or a dose of drinking water vaccine may contain a dose of 30 to 1000 μ l in volume. Preferably, the total volume of a single dose is between about 30. mu.l and 1000. mu.l, more preferably between about 50. mu.l and 500. mu.l, more preferably between about 75. mu.l and 250. mu.l, even more preferably between about 100. mu.l and 200. mu.l, most preferably a single dose of 100. mu.l, 110. mu.l, 120. mu.l, 125. mu.l, 130. mu.l, 135. mu.l, 140. mu.l, 145. mu.l, 150. mu.l, 160. mu.l, 170. mu.l, 175. mu.l, 180. mu.l, 190. mu.l, 155. mu.l or 200. mu.l.
In one aspect of the invention, the immunogenic composition is administered in two or more doses.
However, the immunogenic composition may be administered in two or more doses, the first dose being administered before the second dose (booster).
In a preferred aspect of the two-administration regimen, the first and second doses of the immunogenic composition are administered in the same amount. Preferably, each dose is the preferred amount specified above. Alternative embodiments include other subsequent doses in addition to the first and second dosage regimens. For example, a third, fourth, or fifth dose may be administered in these aspects. Preferably, the subsequent third, fourth and fifth dosage regimens are administered in the same amount as the first dosage and the time frame between doses is identical to the time between the first and second dosages described above.
Preferably, the first administration of the vaccine is performed within three weeks of age, more preferably within one week of age, and most preferably within one day of age by the method described below. The second administration may be performed within 20 weeks of age, preferably within 16-18 weeks of age, more preferably within 6-12 weeks of age. Illustratively, the initial (first) vaccination is performed at 1-10 days of age, and the second vaccination (boost) is performed using live or inactivated vaccine at 6-12 or 16-18 weeks of age. More preferably, the initial (first) vaccination is performed at one day of age and the second vaccination (boost) is performed using live or inactivated vaccine at 6-12 weeks of age or 16-18 weeks of age.
In the case of in ovo vaccines, the first administration is preferably performed when the embryo is at day 15 to 19, preferably at day 17, 18 or 19, most preferably at day 18. The second administration may be performed within the first three weeks after birth, preferably within the first 10 days after birth.
In one aspect of the invention, the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, via spray, via drinking water, or via eye drops.
The immunogenic composition is preferably administered locally or systemically. Suitable routes of administration for routine use are oral or parenteral administration, for example intranasal, intravenous, intradermal, transdermal, intramuscular, intraperitoneal, subcutaneous as well as inhalation, in ovo, by spray, by drinking water or by eye drops. However, depending on the nature and mode of action of the compound, the immunogenic composition may also be administered by other routes. Such other routes include, for example, percutaneous, intravenous, intravascular, intraarterial, intranasal, intrathoracic, intratracheal, intracardiac, intraperitoneal, intramedullary, extraperitoneal, intrarectal, and intravaginal. Most preferably, however, the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, by spraying, by drinking water or by eye drops.
The live IBV vaccine is preferably administered alone via eye drops, intranasally, intramuscularly or subcutaneously.
More preferably, bulk administration methods are used, including drinking water and aerosol vaccination. Also preferred is the use of the vaccine as an embryonic vaccine (so-called in ovo vaccine), as described further below.
For example, broilers may be vaccinated at one day of age or 1-3 weeks of age, particularly for broilers with high levels of MDA. Laying hens or breeder chickens may be initially vaccinated at 1-10 days of age and boosted with vaccine at 7-12 or 16-18 weeks of age.
In ovo administration
As indicated above, the present invention also provides an IBV vaccine which can be safely administered by the in ovo route while being able to induce a protective immune response. In ovo administration is well known to those skilled in the art and can be performed without undue effort by those skilled in the art. In ovo administration of vaccines involves administration of the vaccine to avian embryos contained in eggs (for a review of in ovo vaccination, see: Ricks et al, veterinary Advances 495-515, 1999). The vaccine may be injected into any suitable compartment of the egg (e.g., allantoic fluid, yolk sac, amnion, air cells, or into the embryo), as described in the art (Sharma; am.j.vet.res.451619-1623, 1984). Preferably, the vaccine is administered under the chorioallantoic membrane (air cell) and chorioallantoic membrane.
Preferably, the vaccine is injected into the embryonated eggs at a later stage of embryonic development, usually in the last quarter of the hatching period, preferably 3-4 days before hatching. Preferably at day 15 to day 19, more preferably at day 17, 18 or 19, and most preferably at day 18. Subsequently, the inoculated embryonated eggs are transferred to an incubator for hatching. The process of in ovo administration may be automated using robotic injection processes described in the prior art.
Conventional vaccines, which are commonly used for post-hatch vaccination of poultry, cannot be used for in ovo vaccination, because the late embryos are highly sensitive to most of the vaccine viruses examined. However, international patent application WO 01/64244 discloses that IBV vaccines can be used for in ovo administration, but must be applied at very low doses. Furthermore, Wakenell et al, 1986(am.J.Vet. Res., 47933-938) disclose passaging IB vaccine virus in tissue culture to render the virus non-pathogenic to embryos.
In one aspect of the invention, the immunogenic composition is administered by eye drops.
Typically, live vaccines for post-hatch administration include attenuated IBV at a concentration of 10 per dose1To 108EID50(50% egg infection dose), preferably 10 doses per dose2To 105EID50More preferably 10 per dose2To 104EID50Even more preferably 10 per dose2To 103EID50。
Live vaccines for in ovo administration typically comprise 10 in a volume of 50 to 100. mu.l, preferably 50. mu.l2-107EID50Embryo, preferably 102-103EID50Attenuated IBV of embryos.
Preferably, the immunogenic composition of the invention comprises an IBV of the invention in an amount of about 1 to about 10log per dose10EID (egg infection dose)50Preferably from about 2 to about 8log per dose10 EID50Preferably from about 2 to about 7log per dose10 EID50More preferably from about 2 to about 6log per dose10 EID50Even more preferably from about 2 to about 5log per dose10 EID50Even more preferably from about 2 to about 4log per dose10 EID50Most preferably from about 2 to about 3log per dose10 EID50The amount of (c). More preferably, the immunogenic composition of the invention comprises IBV of the invention in an amount per doseAbout 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or log of the agent10 EID50。
In one aspect of the invention, the immunogenic composition comprises 1 to 10log per dose10IBV of EID 50.
In one aspect of the invention, the immunogenic composition comprises 2 to 5log per dose10IBV of EID 50.
In one aspect of the invention, the immunogenic composition comprises 2-4log per dose10IBV of EID 50.
In one aspect of the invention, the immunogenic composition is administered within one week of age, within three days of age, within two days of age, or within one day of age of the subject.
Preferably, the subject to be immunized is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days old. More preferably, the subject to be immunized is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days old. Most preferably, the subject to be immunized is 1, 2,3, 4, 5, 6 or 7 days old.
However, it must be understood that several days after vaccination of a subject, the immune system of the poultry does take several days to establish immunity against IBV infection. Therefore, it is preferred that the immunization is performed within the first 24 hours after birth of the subject.
In one aspect of the invention, the immunogenic composition is administered within the first day of birth of the subject. As shown in the examples, the immunization compositions provided herein have proven safe and effective when administered to 1 day old poultry.
In one aspect of the invention, the method results in an improvement of an efficacy parameter selected from the group consisting of: preventing or reducing cilia stasis, preventing or reducing rale, preventing or reducing egg drop, preventing or reducing kidney pathology, preventing or reducing watery diarrhea, reducing weight loss, reducing viral load, reducing viral shedding, or a combination thereof.
The term "treatment and/or prevention" has been defined elsewhere, wherein the terms "prevention" and "preventing" or "avoiding" are used interchangeably in this application. In addition, the term "slough" has also been defined elsewhere.
The term "reduce", "decrease" or "lower" means that the efficacy parameter (cilia arrest, rho, egg drop, kidney pathology, watery diarrhea, weight reduction, viral load, virus shedding) is reduced by at least 10%, preferably by at least 20%, more preferably by at least 30%, even more preferably by at least 40%, even more preferably by at least 50%, even more preferably by at least 60%, even more preferably by at least 70%, even more preferably by at least 80%, even more preferably by at least 90%, even more preferably by at least 95% and most preferably by 100% compared to a subject of a non-immune control group of the same species. It is common knowledge of the skilled person how to measure the improvement of efficacy parameters.
The term "viral load" is well known to those skilled in the art. The term viral load is used interchangeably with viral titer herein. Viral load or viral titer is a measure of the severity of active viral infection and can be determined by methods known to those skilled in the art. The assay may be based on the detection of viral proteins, such as by binding of antibodies to viral proteins and further detection, or the detection of viral RNA by amplification methods such as RT-PCR. Monitoring viral RNA associated with viruses in plasma by nucleic acid amplification methods is a widely used parameter to assess the state and progression of retroviral disease, and to assess the effectiveness of prophylactic and therapeutic interventions. For example, viral load or viral titer can be calculated by estimating the number of live virus in the body fluid involved, such as RNA copies per ml of plasma.
The term "ciliary retention" is well known to those skilled in the art. The surface of the trachea is covered with specialized epithelial cells, lined with numerous, mobile, hairy structures called cilia. The term "cilia stasis" includes a reduction or loss of cilia and/or a loss or partial loss of cilia activity. Those skilled in the art can determine cilia stasis without undue effort.
The term "rale" is well known to those skilled in the art. However, the term "rale" includes tracheal rales and refers to sounds emanating from the bronchi. Those skilled in the art can determine the pitch without undue effort.
The term egg drop is well known to those skilled in the art. The term "egg reduction" includes a reduction in egg production.
In one aspect of the invention, the treatment or prevention results in the prevention or reduction of ciliary retention compared to a subject of an untreated control group of the same species.
In one aspect of the invention, the treatment or prevention results in the prevention or reduction of kidney disease compared to a subject of an untreated control group of the same species.
In one aspect of the invention, the treatment or prevention results in the prevention or reduction of egg loss as compared to a subject of an untreated control group of the same species.
The invention further provides an IBV or immunogenic composition as described herein for therapeutic use.
The invention further provides an IBV or immunogenic composition as described herein for use as an immunogen or vaccine.
The invention also provides an IBV or immunogenic composition as described herein for use as a medicament.
The invention further provides the use of an IBV or immunogenic composition as described herein in the manufacture of a medicament.
The invention further provides the use of an IBV or immunogenic composition as described herein in the treatment and/or prevention of an IBV infection in a subject.
The invention further provides an immunogenic composition comprising H52IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or fragment thereof, wherein the H52IBV comprises a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having an amino acid sequence as shown in SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, AY044185(SEQ ID NO 80), AF352310(SEQ ID NO 81), AF317210(SEQ ID NO 82) or AF286185(SEQ ID NO 83) or a sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto, and wherein the heterologous S protein or fragment thereof is selected from 4/91S or a fragment thereof, The genotype or serotype of QX, Q1, Arkansas, variant 2, and Brazil, or an amino acid sequence selected from SEQ ID NO 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto.
In another particular aspect of the immunogenic composition according to the invention, the heterologous S protein is a full-length spike protein.
In another particular aspect of the immunogenic composition according to the invention, the fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1077 amino acids in length starting from the N-terminus.
In another particular aspect of the immunogenic composition according to the invention, the fragment of the heterologous S (spike) protein is the extracellular domain of the spike protein.
In another particular aspect of the immunogenic composition according to the invention, the IBV is attenuated.
The present invention further provides a method of preparing an immunogenic composition for the treatment and/or prevention of IBV infection in a subject, the method comprising:
a.) providing H52IBV comprising a spike (S) protein, a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having the amino acid sequence as set forth in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. AF352315(SEQ ID NO 79), AY044185(SEQ ID NO 80), AF352310(SEQ ID NO 81), AF317210(SEQ ID NO 82) or AF286185(SEQ ID NO:83) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto; and
b.) providing a heterologous S protein or fragment thereof selected from the group consisting of 4/91, QX, Q1, Arkansas, variant 2 and a list of genotypes or serotypes of Brazil, or selected from the group consisting of the amino acid sequences shown in SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto; and
c.) replacing the spike protein or fragment thereof of H52IBV of a) with the heterologous S (spike) protein or fragment thereof of b) to obtain H52IBV having the heterologous S protein or fragment thereof; and
d.) obtaining the H52IBV having a heterologous S protein or fragment thereof; and
e.) adding a pharmaceutically acceptable carrier.
The term "obtaining" includes harvesting, isolating, purifying and/or formulating (e.g., completing, inactivating and/or mixing) said IBV H52 with a heterologous S protein or fragment thereof.
The term "harvesting" refers to collecting or recovering the IBV H52 with a heterologous S protein or fragment thereof from transfected or infected cells or cell lines. Any conventional method known in the art, such as any separation method, may be used. Methods well known in the art include centrifugation or filtration, for example using a semi-permeable membrane having a certain pore size.
The term "isolation" includes the isolation step of said IBV H52 with a heterologous S protein or fragment thereof. Methods for isolation from transfected or infected cells or cell lines are known to those skilled in the art. These methods include physical and/or chemical methods including, but not limited to, freeze-thaw cycles, sonication, and the like.
Methods for "purifying" said IBV H52 with heterologous S protein or fragments thereof from isolates are known to the skilled person, e.g. by in "protein purification methods-a practical method" (e.l.v.harris and s.angel, eds., IRL Press at Oxford University Press). These methods include, but are not limited to, separation by centrifugation and/or filtration, precipitation, size exclusion (gel filtration) chromatography, affinity chromatography, metal chelate chromatography, ion exchange chromatography, covalent chromatography, hydrophobic interaction chromatography, and the like. The carrier can be obtained in pure form, purified or free or substantially free of other cellular material or culture medium, etc. After said isolation and/or purification, the antigen exhibits a purity of at least 80%, preferably 80% to 90%, more preferably 90% to 97%, most preferably more than 97%, up to an absolutely pure form without any contamination.
According to another aspect, "obtaining" as used herein may also include further finishing steps as part of the final formulation process, such as adding a buffer, inactivation, neutralization steps, and the like.
In another particular aspect of the method of making an immunogenic composition according to the invention, the fragment of a heterologous S (spike) protein is the extracellular domain of a spike protein.
In another particular aspect of the immunogenic composition according to the invention, the pharmaceutically acceptable carrier is selected from the group consisting of solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, adjuvants, immunostimulants and combinations thereof.
In another particular aspect of the method of making an immunogenic composition according to the invention, the heterologous S protein is a full-length spike protein.
The invention further relates to a plasmid comprising a nucleic acid encoding part of the H52IBV (infectious bronchitis Virus) genome, including heterologous IBV S (spike) protein or a fragment thereof, such as the pUC57-S H52 rIBV CR 88S Ecto donor plasmid (SEQ ID NO: 21).
Clause and subclause
The following clauses are also described herein:
1. h52IBV (infectious bronchitis Virus) encoding a heterologous IBV S (spike) protein or a fragment thereof.
2. An immunogenic composition comprising H52IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or fragment thereof.
3. An immunogenic composition comprising the IBV (infectious bronchitis virus) of clause 1.
IBV H52-definition of protein coding sequence
4. The IBV or immunogenic composition of any of clauses 1 to 3, wherein H52IBV has or consists of or comprises a nucleotide sequence as set forth in EU817497(SEQ ID NO 78) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
5. The IBV or immunogenic composition of any of clauses 1 to 4, wherein H52IBV has or consists of or comprises a spike (S1) protein having the amino acid sequence shown as AF352315(SEQ ID NO 79) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
An immunogenic composition of IBV or any of clauses 1 to 5 wherein the H52IBV strain has or consists of or comprises a spike (S) protein having the amino acid sequence shown as SEQ ID NO 1 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
7. The IBV or immunogenic composition of any of clauses 1 to 6, wherein H52IBV has or consists of or comprises a nucleocapsid (N) protein having the amino acid sequence shown as AY044185(SEQ ID NO 80) or AF352310(SEQ ID NO 81) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
8. The IBV or immunogenic composition of any of clauses 1 to 7, wherein H52IBV has or consists of or comprises a nucleocapsid (N) protein having the amino acid sequence as set forth in SEQ ID NO:2 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
9. The IBV or immunogenic composition of any of clauses 1 to 8, wherein H52IBV has or consists of or comprises an envelope (E) protein having the amino acid sequence shown as AF317210(SEQ ID NO 82) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
10. The IBV or immunogenic composition of any of clauses 1 to 9, wherein H52IBV has or consists of or comprises an envelope (E) protein having the amino acid sequence shown in SEQ ID No. 3 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
11. The IBV or immunogenic composition of any of clauses 1 to 10, wherein H52IBV has or consists of or comprises a membrane glycoprotein (M) having an amino acid sequence as shown in AF286185(SEQ ID NO 83) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
12. The IBV or immunogenic composition of any of clauses 1-11, wherein H52IBV has or consists of or comprises a membrane glycoprotein (M) protein having the amino acid sequence set forth in SEQ ID No. 4 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto.
Heterologous S protein
13. The IBV or immunogenic composition of any of clauses 1-12, wherein the heterologous spike is a non-Massachusetts genotype or serotype.
14. The IBV or immunogenic composition of any of clauses 1-13, wherein the heterologous S protein or fragment thereof is from an IBV having a genotype or serotype selected from the group consisting of: arkansas (e.g., Arkansas 99), Brazil (e.g., BR-1, BR-2, 23/2013, IBV/Brasil/351/1984), California (e.g., California1734/04, California99), Connecticut, Delaware (e.g., Delaware 98), Dutch (e.g., D207, D212, D274, D3128, D3896, D8880, D1466), Florida, Georgia (e.g., Georgia GA-07, GA-08, GA-12, GA-13), Gray, Holte, Iowa (e.g., Iowa 97 and Iowa 69), Italy (e.g., Italy 02), JMK, IBT 3, Maine (e.g., Mannsylvanine 209), Pennsylvania (e.g., Pennsylvania 1220/98, Pennsylvania/38998), Wolk (e.g., Wolk 084, Wolk 845), Wolk (e.g., CmvlSE/842, such as variants of Cqqqqqv/387), gamma CoV/Ck/Poland/G052/2016) and 4/91(793B, CR 88).
15. The IBV or immunogenic composition of any of clauses 1-14, wherein the heterologous S protein or fragment thereof is from an IBV selected from the group consisting of genotypes or serotypes of: 4/91, QX, Q1, Italy 02, Arkansas, Connetictu, Georgia, LDT3, PL84084, variant 2 or Brazil.
16. The IBV or immunogenic composition of any of clauses 1-15, wherein the heterologous S protein or fragment thereof is from an IBV selected from the group consisting of genotypes or serotypes of: 4/91, QX, Q1, Arkansas, variant 2 and Brazil.
17. The IBV or immunogenic composition of clause 16, wherein the 4/91 strain is selected from the group consisting of: spain/98/328, Spain/92/35, IR-3654-VM, FR-CR88061-88, FR-85131-85, UK-1233-95, UK/3/91, Spain/00/336, UK/7/91, 4/91-pathogenic, 4/91 attenuated, IB4-91 and CR 88.
18. The IBV or immunogenic composition of clause 16, wherein the strain QX is selected from the group consisting of: FR-L1450T-05, FR-L1450L-05, NL-L1449T-04, NL-L1449K-04, IBV/Ck/SP/170/09, IBV/Ck/SP/79/08, IBV/Ck/SP/248/09, HBN, IBVQX, LX4, BJQ, CK/CH/LGD/03 and GB 341/96.
19. The IBV or immunogenic composition of clause 16, wherein strain Q1 is selected from the group consisting of: CK/CH/LDL/98I, CK/CH/LSD/08-10, J2, Q1, AR08ER22, AR08BA21 and Chile-295-10.
20. The IBV or immunogenic composition of clause 16, wherein the Arkansas strain is selected from the group consisting of: ark99, ArkGA, ArkDPI, AL/5364/00, ARKDPI11, AL/0803/01, AL/7149/00, ArkDPI101, AL/1221/01, AL/1793/01 and AL/4614/98.
21. The IBV or immunogenic composition of clause 16, wherein the variant 2 strain is selected from the group consisting of: IS/1494/06, IBV/Ck/EG/CU/4/2014, gamma CoV/Ck/Poland/G052/2016, Eg/CLEVB-2/IBV/012, D1344/2/4/10_ EG, TR8 and IB VAR 2-06.
22. The IBV or immunogenic composition of clause 16, wherein the Brazil strain is selected from BR-1, BR-2, 23/2013 and IBV/Brasil/351/1984.
23. The IBV or immunogenic composition of any of clauses 1-22, wherein the heterologous S protein or fragment thereof is from 4/91 genotype or serotype.
24. The IBV or immunogenic composition of any of clauses 1-23, wherein the heterologous S protein or fragment thereof is from an 4/91 genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity to SEQ ID No. 5 or 6, or the heterologous S protein or fragment thereof comprises or consists of an amino acid sequence set forth in SEQ ID No. 5 or 6 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto.
25. The IBV or immunogenic composition of any of clauses 1-24, wherein the heterologous S protein or fragment thereof is from a QX genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity to SEQ ID No. 7 or 8, or comprises or consists of an amino acid sequence set forth in SEQ ID No. 7 or 8 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto.
26. The IBV or immunogenic composition of any of clauses 1-25, wherein the heterologous S protein or fragment thereof is from a Q1 genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity to SEQ ID No. 9 or 10, or comprises or consists of an amino acid sequence set forth in SEQ ID No. 9 or 10 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto.
27. The IBV or immunogenic composition of any of clauses 1-26, wherein the heterologous S protein or fragment thereof is from an Arkansas genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity to SEQ ID No. 11 or 12, or comprises or consists of an amino acid sequence as set forth in SEQ ID No. 11 or 12 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto.
28. The IBV or immunogenic composition of any of clauses 1-27, wherein the heterologous S protein or fragment thereof is from a variant 2 genotype or serotype that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity to SEQ ID No. 13 or 14, or the heterologous S protein or fragment thereof comprises or consists of an amino acid sequence set forth in SEQ ID No. 13 or 14 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto.
29. The IBV or immunogenic composition of any of clauses 1-28, wherein the heterologous S protein or fragment thereof is from a Brazil genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity to SEQ ID No. 15 or 16, or the heterologous S protein or fragment thereof comprises or consists of an amino acid sequence as set forth in SEQ ID No. 15 or 16 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto.
30. The IBV or immunogenic composition of any one of clauses 1-29, wherein the heterologous S protein or fragment thereof is selected from SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.
31. The IBV or immunogenic composition of any of clauses 1-30, wherein the heterologous S protein is a full-length spike protein.
32. The IBV or immunogenic composition of any one of clauses 1-31, wherein the fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1077 amino acids in length.
33. The IBV or immunogenic composition of any one of clauses 1-32, wherein the fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1077 amino acids in length starting from the N-terminus.
34. The IBV or immunogenic composition of any of clauses 1-33, wherein the fragment of the heterologous S (spike) protein is at least 1000 amino acids in length.
35. The IBV or immunogenic composition of any of clauses 1-34, wherein the heterologous S (spike) protein is the extracellular domain of a spike protein.
36. The IBV or immunogenic composition of any of clauses 1-35, wherein the heterologous S (spike) protein or fragment thereof replaces the homologous S protein or fragment thereof.
37. The IBV or immunogenic composition of any of clauses 1-36, wherein the heterologous S (spike) protein or fragment thereof replaces a naturally occurring S protein or fragment thereof.
38. The IBV or immunogenic composition of any of clauses 1-37, wherein the heterologous S (spike) protein or fragment thereof replaces the S protein of H52 or fragment thereof.
39. The IBV or immunogenic composition of any one of clauses 1-38, wherein the IBV is attenuated.
40. The IBV or immunogenic composition of any one of clauses 1-39, wherein the IBV is inactivated.
41. The IBV or immunogenic composition of any one of clauses 1-40, wherein the IBV is genetically engineered.
42. The IBV or immunogenic composition of any one of clauses 1-41, wherein the IBV is a recombinant IBV.
43. The immunogenic composition of any one of clauses 2-42, wherein the immunogenic composition is a vaccine.
44. The immunogenic composition of any one of clauses 2-43, wherein the immunogenic composition comprises a pharmaceutically acceptable carrier.
45. The immunogenic composition of clause 44, wherein the pharmaceutically acceptable carrier is phosphate buffered saline.
46. The immunogenic composition of any one of clauses 2-45, wherein the immunogenic composition is effective to treat and/or prevent clinical signs caused by IBV in a subject in need thereof.
47. The immunogenic composition of any one of clauses 2-46, wherein the immunogenic composition protects against challenge with an IBV strain of the serotype of the heterologous spike protein.
48. The immunogenic composition of any one of clauses 2-47, wherein the immunogenic composition protects against 4/91, QX, Q1, Arkansas, variant 2, or Brazil genotypic strain.
49. The immunogenic composition of any one of clauses 2-48, wherein the immunogenic composition protects against challenge with the 4/91 genotype strain.
50. The immunogenic composition of any one of clauses 2-49, wherein the immunogenic composition is formulated for single dose administration.
51. The immunogenic composition of any one of clauses 2-50, wherein the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, by spraying, by drinking water, or eye drops.
52. The immunogenic composition of any one of clauses 2-51, wherein the immunogenic composition comprises 1-10log10 EID50IBV/dose.
53. The immunogenic composition of any one of clauses 2-52, wherein the immunogenic composition comprises 2-5log10EID50IBV/dose.
54. The immunogenic composition of any one of clauses 2-53, wherein the immunogenic composition comprises 2-4log10EID50IBV/dose.
55. A kit comprising an IBV or immunogenic composition of any of clauses 1-54.
56. The kit according to clause 55, wherein the kit further comprises instructions for treating and/or preventing an avian disease.
57. The kit according to clause 55, wherein the kit further comprises instructions for treating and/or preventing a disease in poultry.
58. The kit according to clause 55, wherein the kit further comprises instructions for treating and/or preventing IB.
59. A method of immunizing a subject comprising administering to the subject an immunogenic composition according to any one of clauses 2-54.
60. A method of treating or preventing a clinical sign caused by IBV in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the immunogenic composition of any one of clauses 2-54.
61. A method of reducing cilia stasis in a subject in need thereof as compared to an unvaccinated control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any of clauses 2-54.
62. The immunogenic composition of any one of clauses 2-54 for use in a method of immunizing a subject, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition.
63. The immunogenic composition of any one of clauses 2-54 for use in a method of treating or preventing a clinical sign caused by IBV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition.
64. The immunogenic composition of any one of clauses 2-54 for use in a method of reducing cilia stasis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition.
65. The method or use of any one of clauses 59-64, wherein the subject is avian.
66. The method or use of any of clauses 59-65, wherein the subject is poultry.
67. The method or use of any of clauses 59-66, wherein the subject is selected from the group consisting of a chicken, turkey, quail, or pheasant.
68. The method or use of any one of clauses 59-67, wherein the subject is a chicken.
69. The method or use of any one of clauses 59-68, wherein the immunogenic composition is administered once.
70. The method or use of any one of clauses 59-69, wherein the immunogenic composition is administered in two or more doses.
71. The method or use of any one of clauses 59-70, wherein the immunogenic composition is administered subcutaneously, intramuscularly, orally, by in ovo injection, by spraying, by drinking water, or eye drops.
72. The method or use of any one of clauses 59-71, wherein the immunogenic composition is administered by eye drops.
73. The method or use of any of clauses 59-72, wherein the immunogenic composition comprises 1-10log10EID50IBV/dose.
74. The method or use of any of clauses 59-73, wherein the immunogenic composition comprises 2-5log10EID50IBV/dose.
75. The method or use of any of clauses 59-74, wherein the immunogenic composition comprises 2-4log10EID50IBV/dose.
76. The method or use of any one of clauses 59-75, wherein the immunogenic composition is administered to the subject within the first week, within the first three days, within the first two days, or within the first day of the subject's age.
77. The method or use of any one of clauses 59-76, wherein the immunogenic composition is administered to the subject within the first day of the subject's age.
78. The method or use of any one of clauses 59-77, wherein the method results in an improvement in an efficacy parameter selected from the group consisting of: preventing or reducing cilia stasis, preventing or reducing rale, preventing or reducing egg drop, preventing or reducing kidney pathology, preventing or reducing watery diarrhea, preventing or reducing weight loss, reducing viral load, reducing viral shedding, or a combination thereof.
79. The method or use of any of clauses 59-78, wherein the treatment or prevention results in preventing or reducing cilia stasis, as compared to an untreated control subject of the same species.
80. The method or use of any of clauses 59-79, wherein the treatment or prevention results in preventing or reducing kidney pathology as compared to an untreated control subject of the same species.
81. The method or use of any of clauses 59-80, wherein the treatment or prevention results in prevention or reduction of egg drop as compared to an untreated control subject of the same species.
82. The IBV or immunogenic composition of any of clauses 1-54 for therapeutic use.
83. The IBV or immunogenic composition of any of clauses 1-54 for use as an immunogen or vaccine.
84. The IBV or immunogenic composition of any one of clauses 1-54 for use as a medicament.
85. Use of an IBV or immunogenic composition of any of clauses 1-54 in the manufacture of a medicament.
86. Use of an IBV or immunogenic composition of any of clauses 1-54 in the treatment and/or prevention of an IBV infection in a subject.
87. An immunogenic composition comprising H52IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or fragment thereof, wherein the H52IBV comprises a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having or comprising or consisting of the sequence: an amino acid sequence as shown in SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, AY044185(SEQ ID NO 80), AF352310(SEQ ID NO 81), AF317210(SEQ ID NO 82) or AF286185(SEQ ID NO 83) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto, and wherein the heterologous S protein or fragment thereof is selected from the group consisting of the following genotypes or serotypes: 4. 91, QX, Q1, Arkansas, variant 2 and Brazil, or an amino acid sequence selected from the group consisting of: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto, or a heterologous S protein or fragment thereof comprising or consisting of the amino acid sequence: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto.
88. The immunogenic composition of clause 87, wherein the heterologous S protein is a full-length spike protein.
89. The immunogenic composition of clause 87, wherein the fragment of the heterologous S (spike) protein is at least 500, 750, 1000, or 1077 amino acids in length starting from the N-terminus.
90. The immunogenic composition of clause 87 or 89, wherein the fragment of the heterologous S (spike) protein is the extracellular domain of the spike protein.
91. The immunogenic composition of any one of clauses 92-95, wherein the IBV is attenuated.
92. A method of preparing an immunogenic composition for treating and/or preventing IBV infection in a subject, comprising:
a.) providing H52IBV comprising a spike (S) protein, a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having or comprising or consisting of the amino acid sequence: SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. AF352315(SEQ ID NO 79), AY044185(SEQ ID NO 80), AF352310(SEQ ID NO 81), AF317210(SEQ ID NO 82) or AF286185(SEQ ID NO:83), or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto; and
b.) providing a heterologous S protein or a fragment thereof selected from the group consisting of 4/91, QX, Q1, Arkansas, variant 2 and the genotype or serotype of Brazil, or selected from the group consisting of the amino acid sequences shown in SEQ ID NO 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto, or a heterologous S protein or a fragment thereof comprising or consisting of the amino acid sequence shown in SEQ ID NO 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, or 16%, or a sequence having at least 90%, 91%, 92%, 93%, 91%, 92%, 99%, 99., 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity; and
c.) replacing the spike protein of H52IBV of a) with the heterologous S (spike) protein of b) or a fragment thereof, such that H52IBV has a heterologous S protein or a fragment thereof; and
d.) obtaining the H52IBV having a heterologous S protein or fragment thereof; and
e.) adding a pharmaceutically acceptable carrier.
93. The method of clause 92, wherein the fragment of the heterologous S (spike) protein is the extracellular domain of the spike protein.
94. The method of clauses 92 or 93, wherein the pharmaceutically acceptable carrier is selected from the group consisting of solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, adjuvants, immunostimulants, and combinations thereof.
95. The method of any of clauses 92 or 94, wherein the heterologous S protein is a full-length spike protein.
Brief Description of Drawings
FIG. 1 in ovo double arrest kinetics of H52 rIBV CR 88S ecto compared to recombinant wild-type viruses CR88 and H52. Data points represent the average of 5 samples per time point. Error bars indicate standard deviation.
Figure 2 summary of ciliary retention scores the sum of 10 individual scores calculated for 10 rings of one animal is represented by a dot in the figure. The fraction corresponding to maximal ciliary retention was 40, while the corresponding fraction without ciliary retention was 0. Mean and significance were calculated using GraphPad Prism and conventional one-way ANOVA test (p < 0.0007).
FIG. 3 summary of kidney tissue RT-qPCR results. Each avian animal is represented by one data point.
FIG. 4 summary of the RT-PCR results of nasal swab eluates. Each individual bird is represented as a data point.
Figure 5 summary of ciliary retention scores the sum of 10 individual scores calculated for 10 rings of one animal is represented by a dot in the figure. The fraction corresponding to maximal ciliary retention was 40, while the corresponding fraction without ciliary retention was 0. Mean and significance were calculated using GraphPad Prism and conventional one-way ANOVA test (p < 0.0001).
Overview of sequences
1, SEQ ID NO: h52IBV spike (S) protein.
2, SEQ ID NO: h52IBV nucleocapsid (N) protein.
3, SEQ ID NO: h52IBV envelope (E) protein.
4, SEQ ID NO: h52IBV membrane glycoprotein (M).
5 and 6 of SEQ ID NO: a heterologous S protein from 4/91 genotype or serotype, or a fragment thereof.
7 and 8 of SEQ ID NO: a heterologous S protein from a QX genotype or serotype, or a fragment thereof.
9 and 10 of SEQ ID NO: a heterologous S protein from Q1 genotype or serotype, or a fragment thereof.
11 and 12 of SEQ ID NO: a heterologous S protein from an Arkansas genotype or serotype, or a fragment thereof.
13 and 14: a heterologous S protein from a variant 2 genotype or serotype, or a fragment thereof.
15 and 16 of SEQ ID NO: a heterologous S protein from a Brazil genotype or serotype, or a fragment thereof.
17 in SEQ ID NO: IBV CR88 spike protein nucleic acid coding sequence.
18, SEQ ID NO: IBV H52 spike protein ectodomain nucleic acid coding sequence.
19, SEQ ID NO: pUC57-s H52 rIBV donor plasmid.
20, SEQ ID NO: IBV CR88 spike protein ectodomain nucleic acid coding sequence.
21, SEQ ID NO: pUC57-S H52 rIBV CR 88S Ecto donor plasmid.
22 to 77: and (3) a primer.
78, SEQ ID NO: EU817497(H52 IBV nucleotide sequence)
79 in SEQ ID NO: AF352315(H52 IBV S protein amino acid sequence)
80, SEQ ID NO: AY044185(H52 IBV N protein amino acid sequence)
81 of SEQ ID NO: AF352310(H52 IBV N protein amino acid sequence)
82 in SEQ ID NO: AF317210(H52 IBV E protein amino acid sequence)
83 of SEQ ID NO: AF286185(H52 IBV M protein amino acid sequence)
84 and 85: primer and method for producing the same
Examples
The examples set forth below are illustrative of specific embodiments of the present invention. These examples are merely illustrative and should not be construed as limiting the scope or underlying principles of the invention.
Example 1: production of recombinant IBV H52 wherein the coding sequence for the H52 spike protein or the extracellular domain of the spike protein is replaced by the coding sequence for the heterologous spike protein or the extracellular domain of the spike protein
Construction of Donor plasmids
Examples of replacement of the ectodomain of the H52 spike protein with the ectodomain of CR88 are detailed: the IBV CR88 spike protein nucleic acid coding sequence (SEQ ID NO: 17) was synthesized by a commercial supplier. It was used as a template to replace the IBV H52 spike protein ectodomain nucleic acid coding sequence (SEQ ID NO: 18) described in pUC57-S IBV-5-1b-S-SIR-3T donor plasmid by van Berrden et al (Virol J.2017; 14 (1): 109), hereinafter referred to as pUC57-S H52 rIBV donor plasmid (SEQ ID NO: 19). SEQ ID NO: bases 1717 to 4941 of 19 are replaced by the corresponding IBV CR88 spike protein ectodomain nucleic acid coding sequence (SEQ ID NO: 20) which corresponds to bases 55 to 3285 of SEQ ID NO 15, respectively. This generated pUC57-S H52 rIBV CR 88S Ecto donor plasmid (SEQ ID NO:21) in which the extracellular domain of the IBV CR88 spike protein is encoded by bases 1717 to 4947. To this end, the pUC57-s H52 rIBV donor plasmid (SEQ ID NO: 19) was digested using unique restriction sites 5'(EcoRV) and 3' (PmlI) near the H52 spike protein coding sequence to linearize the plasmid and remove the H52 spike protein and flanking sequences. A QIAquick gel extraction kit (Qiagen) was used to purify the band corresponding to the pUC57-s IBV H52 backbone without the H52 spike protein coding sequence. By using
Figure BDA0003043693370000331
High fidelity DNA polymerase (NEB; see Table 1 for primers) amplified the CR88 spike protein ectodomain nucleic acid coding sequence and flanking 5 'and 3' IBV H52 sequences in three separate PCR reactions. The PCR products were purified by QIAquick gel extraction (Qiagen) and used for Gibson assembly with hibaid DNA assembly cloning kit (NEB) according to the kit protocol to generate pUC57-S H52 ribbv CR 88S eco donor plasmid.
Table 1 Gibbson assembly primers designed by NEB using the NEBuilder in-line tool for generating PCR products to assemble pUC57-S H52 rIBV CR 88S Ecto donor plasmid.
Figure BDA0003043693370000332
Targeted RNA recombination and rescue of recombinant IBV
For the generation of recombinant IBV, a method of targeted RNA recombination as described by van Berrden et al (Virol J.2017; 14 (1): 109) was employed. Briefly, H52 murine (m) IBV was generated as described. To generate H52 rIBV CR 88S Ecto, LR7 cells were infected with H52 mIBV and electroporated with in vitro transcripts generated from pUC57-S H52 rIBV CR 88S Ecto donor plasmids, which were then injected into 8-day-old embryonated SPF eggs (VALO BioMedia). After incubation at 37.5 ℃ and 60% humidity for 9 days, the cells were purified by QIAamp Virus RNA mini kit (Qiagen) and used with PlatinumTMSuperScript of Taq DNA polymerase (ThermoFisher)TMIII after RNA isolation by a one-step RT-PCR system, allantoic fluids of all eggs were analyzed separately to rescue recombinant IBV. Primers PO1323 and PO1729 (Table 2) bound to the extracellular domains of H52IBV 1ab and CR88 IBV S were used, which were specific for recombinant IBV but not mIBV. Allantoic fluid inoculated with the highest dilution of LR7 cells was selected for two rounds of end-point dilution in 8 day old SPF eggs. Magmaxtm core nucleic acid purification kit (ThermoFisher) and KingFisher were usedTMThe Duo Prime purification system (ThermoFisher) performs nucleic acid isolation of limiting dilution samples, followed by analysis for the presence of rIBV by RT-PCR as described above. After the second limiting dilution, the test positive allantoic fluid of eggs inoculated at the highest dilution was used for propagation in 10 day old embryonated SPF eggs. Allantoic fluid was diluted in 1xPBS at a ratio of 1: 1000 dilution and 100. mu.l per egg. Allantoic fluid was harvested 48 hours after inoculation, cleared of debris and stored at-80 ℃. To confirm the sequence from the donor plasmid in the resulting rIBV, viral nucleic acids were isolated using the QIAamp viral RNA mini kit, followed by SuperScript III one-step RT-PCR using the primers listed in Table 3, QIAquick PCR purification and subsequent procedures, Sanger sequencing using the same primers, by a commercial supplier.
TABLE 2 primers for identifying recombinant IBV after rescue
Figure BDA0003043693370000341
Table 3 sequencing primers to confirm the sequence of the donor region in H52 rIBV CR 88S Ecto
Figure BDA0003043693370000342
Generation and characterization of H52 recombinant IBV in which the coding sequence for the H52 spike protein or the ectodomain of the spike protein is replaced by the coding sequence for the spike protein or the ectodomain of the spike protein of another IBV genotype
Recombinant H52IBV was generated and characterized using the same method as described for the generation of H52 rIBV CR 88S Ecto, wherein the spike protein coding sequence (bases 1663 to 5151 of SEQ ID NO: 19) or the H52 spike protein ectodomain coding sequence (bases 1717 to 4941 of SEQ ID NO: 19) was replaced with the coding sequence for the IBV spike protein or spike protein ectodomain of the serotypes and genotypes as listed in Table 4.
TABLE 4 Gibson assembled primers for H52 rIBV donor plasmid with heterologous spike protein or extracellular domain of spike protein
Figure BDA0003043693370000343
Figure BDA0003043693370000351
Figure BDA0003043693370000361
The primers in tables 2 and 3 were used to identify and sequence different recombinant viruses and were modified as necessary for the corresponding spike protein sequences.
Example 2: in ovo multiple braking mechanics
Is eight daysAged embryonated eggs were inoculated with 102EID50And a separate control. Eggs were incubated at 37.5 ℃ and 60% humidity and were candled once daily after 0, 8, 24, 34, 48 and 72 hours of incubation and embryo mortality was recorded. Five preselected eggs were taken for each sample and time point and transferred to 4 ℃ for at least 2 hours. Subsequently, allantoic fluid was collected and stored at-80 ℃. For analysis, samples were thawed and diluted 1:10 in 1xPBS without Ca and Mg, nucleic acids were extracted using QIAamp DNA Blood Mini kit (Qiagen), and vector RNA was added using a Hamilton Starlet automated pipettor. The relative amount of IBV RNA of the extracted nucleic acids was analyzed by RT-qPCR, adapted using the protocol of Callison et al (J Virol methods.2006; 138(1-2): 60-5). Briefly, the same primers and probes were used, and the temperature protocol was adjusted to suit the application
Figure BDA0003043693370000362
Fast Virus 1-Step Master Mix (ThermoFisher) and ABITM7900HT Fast real-time PCR System (Thermo Fisher Scientific). All nucleic acid samples were analyzed in triplicate, using 10-fold serial dilutions of IBV H52 as reference.
Slightly similar replication kinetics were observed for H52 rlbv CR 88S ecoto at early time points compared to recombinant wild-type viruses H52 and CR 88. However, after 32 hours, all viruses reached comparable ct values. All embryos survived 32 hours post inoculation, while at the 48 hour post infection time point all remaining embryos of all samples died. Thus, replication of H52 rlbv CR88 sector was considered equally efficient compared to the wild-type virus (see figure 1).
Example 3: preparation of vaccines and challenge viruses
To confirm the efficacy of H52 rIBV with heterologous spike protein or extracellular domain of spike protein in chickens, equal portions of the virus stock were thawed, diluted 10-fold in 1xPBS, and 50% Embryo Infection Dose (EID) was determined by inoculating 100. mu.l of each dilution into 5 8-day-old embryonated chicken eggs50). The eggs were incubated at 37.5 ℃ and 60% humidity until 7 days after inoculation. Will have death after 24 hoursEggs with dead embryos were excluded from this experiment. All other eggs with dead embryos 7 days after inoculation were considered positive. On day 7 post inoculation, all eggs with live embryos were candled from the bottom to identify dwarf chicks that were considered positive. Calculating EID by using formulas of Reed and Muench50Perml (Am J epidemic, 1938; 27(3):493 497). For immunization, the virus stock was diluted in 1xPBS to obtain 104.3EID50Titer per ml (10 per chicken in 50. mu.l)3EID50)。
The challenge viruses of genotype and serotype 793B, QX, Q1, Ark, variant 2, and Brazil were propagated in 10-day-old embryonated SPF eggs. 24 hours after inoculation, the eggs were transferred to 4 ℃ for at least 2 hours. Allantoic fluid was collected, aliquoted and stored at-80 ℃. Viral titers were determined as described above. Titer was set to 10 by dilution with 1xPBS4.3To 105.3 EID50Perml (10 per chicken in 50. mu.l)3To 104EID50)。
Example 4: determination of vaccine efficacy
Fertilized SPF eggs were incubated in an egg incubator for 18 days at 99.7 ° F and 50% humidity with one rotation per hour. On day 18 of incubation, eggs were candled and fertilized eggs were transferred to an incubator and incubated at 99 ° F and 70% humidity until hatch. Chickens without clinical signs or deformities were randomly assigned to each treatment group and moved to individual isolators. At least two chickens were used as a Stringent Negative Control (SNC) group, five were included in the Challenge Control (CC) group, and at least ten chickens per group were inoculated with recombinant IBV having a heterologous spike protein or extracellular domain of a spike protein, followed by challenge. Animals were kept under feeding conditions that met local and national recommendations for animal welfare. The lighting schedule was adjusted to 16 hours per day. Feed and water were provided ad libitum. After transfer to the isolator, the chickens (1 day old) were inoculated with eye drops (total volume 50. mu.l, 25. mu.l per eye) 103EID50However, the SNC and CC groups were not treated. 21 days after inoculation, the chickens in the CC group and the vaccinated group were administered 10 times by eye drops (total volume 50. mu.l, 25. mu.l per eye)3To 104EID50Respective spike-homologous challenge strains (793B, QX, Q1, Ark, variant 2 or Brazil) were challenged. On day 7 post challenge, all chickens were euthanized, throat swabs removed, kidneys removed, and stored in RNAlater stable (ThermoFisher) at 4 ℃ for IBV-specific RT-qPCR analysis. In addition, the trachea was removed and transferred to a 50ml tube containing warmed cell culture medium. The trachea is then cleared from the connective tissue and rinsed with cell culture medium. The trachea was cut into tracheal rings using a McIlwain tissue chopper set at a slice thickness of 0.6-0.8 mm. Each of the three rings in the upper, four rings in the middle and three rings in the lower part of the trachea were analyzed by light microscopy and scored for ciliary retention (see table 5). Normal is recorded if more than 50% of the inner rings show significant ciliary movement (fraction 2 and below). If less than 50% of the cilia move (fractions 3 and 4), the ring is recorded as positive for cilia stasis.
For IBV-specific RT-qPCR analysis, kidney tissue sections were warmed to room temperature and then transferred to separate 2ml precell tubes filled with medium and PBS, respectively. By using
Figure BDA0003043693370000371
Tissue homogenizers (Bertin Instruments) homogenize kidneys at 6800rpm for 1 × 20 seconds. Throat swabs were eluted with 2ml of 1 xPBS. Using MagMAXTMCore nucleic acid purification kit (ThermoFisher) and KingFisherTMThe Duo Prime purification system (ThermoFisher) separated nucleic acids from 200. mu.l of the eluate and tissue homogenate, respectively. Except that StepOnePlus was usedTMReal-time PCR system (ThermoFisher) RT-qPCR was performed as described above for in ovo kinetics, except that analysis was performed in duplicate.
Table 5: ciliary arrest score in tracheal rings
Ciliary activity [% ]] Ciliary retention score
100 0
<100-75 1
<75-50 2
<50-25 3
<25-0 4
Example 5: potency of recombinant IBV H52 encoding heterologous spike proteins or extracellular domains of spike proteins
The purpose of this study was to demonstrate that vaccination with recombinant IBV H52(Mass genotype) encoding a heterologous spike protein or the ectodomain of a spike protein can confer protection against challenge with a homologous challenge strain of the spike protein.
Studies have analyzed whether recombinant IBV H52(Mass genotype) encoding the extracellular domain of the spike protein of IBV CR88(4/91 genotype) is able to confer protection against challenge with the virulent 793B strain (4/91 genotype), which is considered to be a homologous attacker of the encoded extracellular domain of the spike protein of IBV CR88, a heterologous attacker of the IBV H52 backbone. All chickens were observed daily for clinical signs. No clinical signs were recorded after vaccination or challenge. The titers of challenge virus with 793B at day 21 post-inoculation were determined to be 10 each by back titration with H52 rIBV CR 88S Etco at 1 day of age4.13EID50Per ml (target 10)4.3EID50Per ml), and 104.69EID50Per ml (target 10)4.3EID50In ml). Move the cilia as described aboveThe stasis was scored and the results are shown in figure 2 and summarized in table 6.
Table 6: summary of ciliary retention protection scores 28 days post-vaccination and 7 days post challenge. The average ciliary retention score for each group (highest score 40, lowest score 0) was calculated by summing the total score for each chicken in each group and dividing the total group score by the number of animals. The animal was considered unaffected if not less than 9 of the 10 loops showed normal ciliary activity.
Figure BDA0003043693370000381
All animals in the strict negative control group showed normal ciliary movement, while 80% of the animals in the challenge control group showed positive ciliary retention. In contrast, 82% of the animals vaccinated with H52 rlbv CR 88S Etco were protected. Furthermore, animals vaccinated with H52 rlbv CR 88S Etco had a reduced viral load in kidney and nasal swabs compared to animals challenged with the control group (fig. 3 and 4).
Furthermore, it was analyzed whether recombinant IBV H52 encoding the IBV QX spike protein could confer protection against challenge with the virulent D388 QX strain, which is considered to be a homologous attacker of the encoded IBV QX spike protein, and a heterologous attacker of the IBV H52 backbone. All chickens were observed daily for clinical signs. No clinical signs were recorded after vaccination or challenge. Titer of 10 determined by reverse titration with CH52 rIBV QX S inoculation at 1 day of age4EID50Per ml, whereas QX vaccination exceeds 105EID50Per ml (target 10)4.3EID50In ml). Titer of 10 determined 21 days post inoculation using D388 QX challenge virus4.83EID50/ml(target 104.3EID50In ml). Cilia stasis was scored as described above and the results are shown in figure 5 and summarized in table 7.
Table 7: an overview of ciliary retention protection scores 28 days post-vaccination and 7 days post challenge. The average ciliary retention score for each group (highest score 40, lowest score 0) was calculated by summing the total score for each chicken in each group and dividing the total group score by the number of animals. The animal was considered unaffected if not less than 9 of the 10 loops showed normal ciliary activity.
Group of Vaccine Attack of Mean ciliary retention score Without affecting [% ]]
1 - - 0 100
2 - D388 QX 38.4 0
3 QX vaccine D388 QX 3.5 100
3 H52 rIBV QX S D388 QX 5.9 100
All animals in the strict negative control group showed normal ciliary movement, while all animals in the challenge control group were positive for ciliary retention. In contrast, animals vaccinated with H52 rlbv QX S or QX vaccine were 100% protected.
Similar results were obtained with other H52 rlbv having heterologous spike proteins or extracellular domains of spike proteins.
This result highlights the suitability of IBV 4/91 as an efficient scaffold for the production of recombinant IBV with heterologous spike proteins, particularly showing excellent results when compared to the data for the prior art IBV Beaudette scaffold.
Sequence listing
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Asn Val Thr Glu Ala Thr Ala Asn Tyr Ser Tyr Leu Ala Asp Gly Gly
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Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln
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Gly Ala Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val
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Ser His Asn Glu Thr Gly Ser Glu Ser Ile Glu Asn Gln Phe Tyr Ile
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Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro
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Asp Gly Ser Leu Ser Ile Ile Val Pro Gln Glu Leu Glu Gln Phe Val
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Ala Pro Leu Phe Asn Val Thr Glu His Val Leu Ile Pro Asp Ser Phe
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Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val
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Gln Ile Ile Cys Leu Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys Arg
610 615 620
Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val
625 630 635 640
Val Asn Gly Val Gly Gln Arg Glu Asp Met Glu Leu Leu Ser Phe Tyr
645 650 655
Ser Ser Thr Lys Pro Ser Gly Tyr Asn Thr Pro Ile Phe Asn Asn Val
660 665 670
Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Leu Thr Pro Pro Asn Ser
675 680 685
Pro Thr Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu
690 695 700
Ser Val Gly Leu Pro Thr Asp Glu Glu Tyr Lys Lys Cys Thr Ala Gly
705 710 715 720
Pro Leu Gly Phe Val Lys Asp Leu Val Cys Ala Arg Glu Tyr Asn Gly
725 730 735
Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Met Tyr
740 745 750
Thr Ser Ser Leu Val Ala Ser Met Ala Leu Gly Gly Ile Thr Ala Ala
755 760 765
Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu
770 775 780
Gly Ile Thr Asn Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala
785 790 795 800
Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Lys Ser Thr
805 810 815
Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ser
820 825 830
Ile Leu Thr Glu Thr Met Gln Ser Leu Asn Lys Asn Phe Gly Ala Ile
835 840 845
Ser Ser Val Ile Gln Asp Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala
850 855 860
Asp Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser
865 870 875 880
Val Leu Ala Ser Ala Lys Gln Ala Glu Tyr His Arg Val Ser Gln Gln
885 890 895
Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser
900 905 910
Asn Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro
915 920 925
Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro
930 935 940
Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Asn Pro
945 950 955 960
Ala Asn Ala Ser His Tyr Ala Ile Val Pro Val Asn Gly Arg Gly Val
965 970 975
Phe Ile Glu Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr
980 985 990
Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys
995 1000 1005
Gln Ala Asn Tyr Val Asn Val Asn Lys Thr Val Ile Asn Thr Phe
1010 1015 1020
Val Glu Asp Asp Asp Phe Asp Phe Tyr Asp Glu Leu Ser Lys Trp
1025 1030 1035
Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Glu Phe Asn
1040 1045 1050
Tyr Thr Val Pro Val Leu Asn Ile Ser Asn Glu Ile Asp Arg Ile
1055 1060 1065
Gln Gln Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu
1070 1075 1080
Thr Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val
1085 1090 1095
Trp Leu Ala Ile Ala Phe Leu Thr Ile Ile Phe Ile Leu Val Leu
1100 1105 1110
Cys Trp Ile Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly
1115 1120 1125
Cys Phe Gly Ile Ile Pro Leu Met Ser Lys Cys Gly Lys Lys Ser
1130 1135 1140
Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val Thr
1145 1150 1155
<210> 6
<211> 1077
<212> PRT
<213> avian infectious bronchitis Virus
<400> 6
Leu Leu Tyr Asp Asn Asn Thr Tyr Val Tyr Tyr Tyr Gln Ser Ala Phe
1 5 10 15
Arg Pro Gly Pro Gly Trp His Leu Tyr Gly Gly Ala Tyr Ala Val Asp
20 25 30
Arg Val Phe Asn Glu Thr Asn Asn Ala Gly Ser Ala Ser Asp Cys Thr
35 40 45
Ala Gly Thr Phe Tyr Glu Ser His Asn Ile Ser Ala Ser Ser Val Ala
50 55 60
Met Thr Val Pro His Asn Gly Met Ser Trp Ser Ala Ser Gln Phe Cys
65 70 75 80
Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr His Cys
85 90 95
Phe Lys Asn Gln Leu Gly Ser Cys Pro Leu Thr Gly Met Ile Pro Gln
100 105 110
Asn His Ile Arg Ile Ser Ala Met Arg Asp Gly Val Leu Phe Tyr Asn
115 120 125
Leu Thr Val Ser Val Ser Lys Tyr Pro Arg Phe Lys Ser Leu Gln Cys
130 135 140
Val Ser Asn Ser Thr Ser Val Tyr Val Asn Gly Asp Leu Val Phe Thr
145 150 155 160
Ser Asn Glu Thr Ser Tyr Val Thr Gly Ala Gly Val Tyr Phe Lys Ser
165 170 175
Gly Gly Pro Val Thr Tyr Lys Val Met Lys Glu Val Lys Ala Leu Ala
180 185 190
Tyr Phe Ile Asn Gly Thr Ala Gln Glu Val Ile Leu Cys Asp Asn Ser
195 200 205
Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp
210 215 220
Gly Phe Tyr Pro Phe Thr Asn His Ser Leu Val Lys Asp Arg Phe Ile
225 230 235 240
Val Tyr Arg Glu Ser Ser Thr Asn Thr Thr Leu Lys Leu Thr Asn Phe
245 250 255
Ser Phe Thr Asn Val Ser Asn Ala Ser Pro Asn Ser Gly Gly Val Asp
260 265 270
Thr Phe Gln Leu Tyr Gln Thr Ser Thr Ala Gln Asp Gly Tyr Tyr Asn
275 280 285
Phe Asn Leu Ser Phe Leu Ser Ser Phe Val Tyr Lys Pro Ser Asp Phe
290 295 300
Met Tyr Gly Ser Tyr His Pro His Cys Lys Phe Arg Pro Glu Asn Ile
305 310 315 320
Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Thr Tyr Gly
325 330 335
Pro Ile Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Asn Arg Ala Thr
340 345 350
Cys Cys Tyr Ala Tyr Ser Tyr Gln Gly Pro Ser Arg Cys Lys Gly Val
355 360 365
Tyr Arg Gly Glu Leu Thr Gln Tyr Phe Glu Cys Gly Leu Leu Val Tyr
370 375 380
Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Ser Glu Pro Leu
385 390 395 400
Val Leu Thr Gln Tyr Asn Tyr Asn Asn Ile Thr Leu Asn Lys Cys Val
405 410 415
Glu Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Phe Ile Thr Asn Val
420 425 430
Thr Glu Ala Thr Ala Asn Tyr Ser Tyr Leu Ala Asp Gly Gly Leu Ala
435 440 445
Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln Gly Ala
450 455 460
Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln
465 470 475 480
Gln Phe Val Val Ser Gly Gly Asn Leu Val Gly Ile Leu Thr Ser His
485 490 495
Asn Glu Thr Gly Ser Glu Ser Ile Glu Asn Gln Phe Tyr Ile Lys Leu
500 505 510
Thr Asn Gly Thr Arg Arg Ser Arg Arg Ser Val Thr Gly Asn Val Thr
515 520 525
Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly
530 535 540
Ser Leu Ser Ile Ile Val Pro Gln Glu Leu Glu Gln Phe Val Ala Pro
545 550 555 560
Leu Phe Asn Val Thr Glu His Val Leu Ile Pro Asp Ser Phe Asn Leu
565 570 575
Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile
580 585 590
Ile Cys Leu Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys Arg Lys Leu
595 600 605
Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn
610 615 620
Gly Val Gly Gln Arg Glu Asp Met Glu Leu Leu Ser Phe Tyr Ser Ser
625 630 635 640
Thr Lys Pro Ser Gly Tyr Asn Thr Pro Ile Phe Asn Asn Val Ser Thr
645 650 655
Gly Asp Phe Asn Ile Ser Leu Leu Leu Thr Pro Pro Asn Ser Pro Thr
660 665 670
Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val
675 680 685
Gly Leu Pro Thr Asp Glu Glu Tyr Lys Lys Cys Thr Ala Gly Pro Leu
690 695 700
Gly Phe Val Lys Asp Leu Val Cys Ala Arg Glu Tyr Asn Gly Leu Leu
705 710 715 720
Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Met Tyr Thr Ser
725 730 735
Ser Leu Val Ala Ser Met Ala Leu Gly Gly Ile Thr Ala Ala Gly Ala
740 745 750
Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile
755 760 765
Thr Asn Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe
770 775 780
Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Lys Ser Thr Ser Leu
785 790 795 800
Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ser Ile Leu
805 810 815
Thr Glu Thr Met Gln Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser
820 825 830
Val Ile Gln Asp Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asp Ala
835 840 845
Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu
850 855 860
Ala Ser Ala Lys Gln Ala Glu Tyr His Arg Val Ser Gln Gln Arg Glu
865 870 875 880
Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Asn Arg
885 890 895
Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn
900 905 910
Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro Glu Ser
915 920 925
Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Asn Pro Ala Asn
930 935 940
Ala Ser His Tyr Ala Ile Val Pro Val Asn Gly Arg Gly Val Phe Ile
945 950 955 960
Glu Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro
965 970 975
Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Ala
980 985 990
Asn Tyr Val Asn Val Asn Lys Thr Val Ile Asn Thr Phe Val Glu Asp
995 1000 1005
Asp Asp Phe Asp Phe Tyr Asp Glu Leu Ser Lys Trp Trp Asn Asp
1010 1015 1020
Thr Lys His Glu Leu Pro Asp Phe Asp Glu Phe Asn Tyr Thr Val
1025 1030 1035
Pro Val Leu Asn Ile Ser Asn Glu Ile Asp Arg Ile Gln Gln Val
1040 1045 1050
Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Thr Leu Ser
1055 1060 1065
Ile Leu Lys Thr Tyr Ile Lys Trp Pro
1070 1075
<210> 7
<211> 1156
<212> PRT
<213> avian infectious bronchitis Virus
<400> 7
Met Leu Val Lys Ser Leu Phe Leu Val Thr Ile Leu Cys Ala Leu Cys
1 5 10 15
Ser Ala Asn Leu Phe Asp Ser Asp Asn Asn Tyr Val Tyr Tyr Tyr Gln
20 25 30
Ser Ala Phe Arg Pro Pro Asn Gly Trp His Leu Gln Gly Gly Ala Tyr
35 40 45
Ala Val Val Ser Ser Thr Asn Tyr Thr Asn Asn Ala Gly Ser Ala His
50 55 60
Gly Cys Thr Val Gly Val Ile Lys Asp Val Tyr Asn Gln Ser Val Ala
65 70 75 80
Ser Ile Ala Met Thr Ala Pro Leu Gln Gly Met Ala Trp Ser Lys Ser
85 90 95
Gln Phe Cys Ser Ala His Cys Asn Phe Ser Glu Ile Thr Val Phe Val
100 105 110
Thr His Cys Tyr Ser Ser Gly Ser Gly Ser Cys Pro Ile Thr Gly Met
115 120 125
Ile Pro Arg Asp His Ile Arg Ile Ser Ala Met Lys Asn Gly Ser Leu
130 135 140
Phe Tyr Asn Leu Thr Val Ser Val Ser Lys Tyr Pro Asn Phe Lys Ser
145 150 155 160
Phe Gln Cys Val Asn Asn Phe Thr Ser Val Tyr Leu Asn Gly Asp Leu
165 170 175
Val Phe Thr Ser Asn Lys Thr Thr Asp Val Thr Ser Ala Gly Val Tyr
180 185 190
Phe Lys Ala Gly Gly Pro Val Asn Tyr Ser Ile Met Lys Glu Phe Lys
195 200 205
Val Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Val Leu Cys
210 215 220
Asp Asn Ser Pro Lys Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn
225 230 235 240
Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Ser Thr Leu Val Arg Glu
245 250 255
Lys Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Ala Leu
260 265 270
Thr Asn Phe Thr Phe Ser Asn Val Ser Asn Ala Gln Pro Asn Ser Gly
275 280 285
Gly Val Asn Thr Phe His Leu Tyr Gln Thr Gln Thr Ala Gln Ser Gly
290 295 300
Tyr Tyr Asn Phe Asn Leu Ser Phe Leu Ser Gln Phe Val Tyr Lys Ala
305 310 315 320
Ser Asp Phe Met Tyr Gly Ser Tyr His Pro Ser Cys Ser Phe Arg Pro
325 330 335
Glu Thr Ile Asn Ser Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu
340 345 350
Thr Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly
355 360 365
Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Arg Gly Pro Met Ala Cys
370 375 380
Lys Gly Val Tyr Ser Gly Glu Leu Ser Thr Asn Phe Glu Cys Gly Leu
385 390 395 400
Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Thr
405 410 415
Glu Pro Leu Val Leu Thr Gln Tyr Asn Tyr Asn Asn Ile Thr Leu Asp
420 425 430
Lys Cys Val Ala Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Phe Ile
435 440 445
Thr Asn Val Thr Asp Ser Ala Ala Asn Phe Ser Tyr Leu Ala Asp Gly
450 455 460
Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Val Phe Val Val
465 470 475 480
Gln Gly Ile Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp
485 490 495
Val Asn Gln Gln Phe Val Val Ser Gly Gly Asn Ile Val Gly Ile Leu
500 505 510
Thr Ser Arg Asn Glu Thr Gly Ser Glu Gln Val Glu Asn Gln Phe Tyr
515 520 525
Val Lys Leu Thr Asn Ser Ser His Arg Arg Lys Arg Ser Ile Gly Gln
530 535 540
Asn Val Thr Ser Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys Ile Glu
545 550 555 560
Pro Asp Gly Ser Leu Lys Met Ile Val Pro Glu Glu Leu Lys Gln Phe
565 570 575
Val Ala Pro Leu Leu Asn Ile Thr Glu Ser Val Leu Ile Pro Asn Ser
580 585 590
Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys
595 600 605
Val Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Leu Glu Cys
610 615 620
Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser
625 630 635 640
Val Val Asn Ser Val Ser Gln Lys Glu Asp Met Glu Leu Leu Ser Phe
645 650 655
Tyr Ser Ser Thr Lys Pro Lys Gly Tyr Asp Thr Pro Val Leu Ser Asn
660 665 670
Val Ser Thr Gly Glu Phe Asn Ile Ser Leu Leu Leu Lys Pro Pro Ser
675 680 685
Ser Pro Ser Gly Arg Ser Phe Ile Glu Glu Leu Leu Phe Thr Ser Val
690 695 700
Glu Thr Val Gly Leu Pro Thr Asp Ala Glu Tyr Lys Lys Cys Thr Ala
705 710 715 720
Gly Pro Leu Gly Thr Leu Lys Asp Leu Ile Cys Ala Arg Glu Tyr Asn
725 730 735
Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr Met
740 745 750
Tyr Thr Ala Ser Leu Val Gly Ala Met Ala Phe Gly Gly Ile Thr Ser
755 760 765
Ala Ala Ala Ile Pro Phe Ala Thr Gln Ile Gln Ala Arg Ile Asn His
770 775 780
Leu Gly Ile Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys Ile Ala
785 790 795 800
Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser
805 810 815
Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser
820 825 830
Ala Ile Leu Thr Glu Thr Met Asn Ser Leu Asn Lys Asn Phe Gly Ala
835 840 845
Ile Thr Ser Val Ile Gln Asp Ile Tyr Ala Gln Leu Asp Ala Ile Gln
850 855 860
Ala Asp Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu
865 870 875 880
Ser Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile Arg Val Ser Gln
885 890 895
Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln
900 905 910
Ser Asn Arg Tyr Gly Phe Cys Gly Ser Gly Arg His Val Leu Ser Ile
915 920 925
Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr
930 935 940
Pro Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Asn
945 950 955 960
Pro Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly
965 970 975
Ile Phe Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr Ala Arg Asp Met
980 985 990
Tyr Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser
995 1000 1005
Cys Gln Ala Asn Tyr Val Asn Val Asn Lys Thr Val Ile Thr Thr
1010 1015 1020
Phe Val Glu Asp Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys
1025 1030 1035
Trp Trp Asn Asp Thr Lys His Gln Leu Pro Asp Phe Asp Asp Phe
1040 1045 1050
Asn Tyr Thr Val Pro Ile Leu Asn Ile Ser Gly Glu Ile Asp Tyr
1055 1060 1065
Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu
1070 1075 1080
Glu Glu Leu Ser Ile Ile Lys Thr Tyr Ile Lys Trp Pro Trp Tyr
1085 1090 1095
Val Trp Leu Ala Ile Phe Phe Ala Ile Ile Ile Phe Ile Leu Ile
1100 1105 1110
Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys
1115 1120 1125
Gly Cys Phe Gly Ile Ile Pro Leu Met Ser Lys Cys Gly Lys Lys
1130 1135 1140
Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val Thr
1145 1150 1155
<210> 8
<211> 1078
<212> PRT
<213> avian infectious bronchitis Virus
<400> 8
Asn Leu Phe Asp Ser Asp Asn Asn Tyr Val Tyr Tyr Tyr Gln Ser Ala
1 5 10 15
Phe Arg Pro Pro Asn Gly Trp His Leu Gln Gly Gly Ala Tyr Ala Val
20 25 30
Val Ser Ser Thr Asn Tyr Thr Asn Asn Ala Gly Ser Ala His Gly Cys
35 40 45
Thr Val Gly Val Ile Lys Asp Val Tyr Asn Gln Ser Val Ala Ser Ile
50 55 60
Ala Met Thr Ala Pro Leu Gln Gly Met Ala Trp Ser Lys Ser Gln Phe
65 70 75 80
Cys Ser Ala His Cys Asn Phe Ser Glu Ile Thr Val Phe Val Thr His
85 90 95
Cys Tyr Ser Ser Gly Ser Gly Ser Cys Pro Ile Thr Gly Met Ile Pro
100 105 110
Arg Asp His Ile Arg Ile Ser Ala Met Lys Asn Gly Ser Leu Phe Tyr
115 120 125
Asn Leu Thr Val Ser Val Ser Lys Tyr Pro Asn Phe Lys Ser Phe Gln
130 135 140
Cys Val Asn Asn Phe Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe
145 150 155 160
Thr Ser Asn Lys Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys
165 170 175
Ala Gly Gly Pro Val Asn Tyr Ser Ile Met Lys Glu Phe Lys Val Leu
180 185 190
Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Val Leu Cys Asp Asn
195 200 205
Ser Pro Lys Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser
210 215 220
Asp Gly Phe Tyr Pro Phe Thr Asn Ser Thr Leu Val Arg Glu Lys Phe
225 230 235 240
Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Ala Leu Thr Asn
245 250 255
Phe Thr Phe Ser Asn Val Ser Asn Ala Gln Pro Asn Ser Gly Gly Val
260 265 270
Asn Thr Phe His Leu Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr
275 280 285
Asn Phe Asn Leu Ser Phe Leu Ser Gln Phe Val Tyr Lys Ala Ser Asp
290 295 300
Phe Met Tyr Gly Ser Tyr His Pro Ser Cys Ser Phe Arg Pro Glu Thr
305 310 315 320
Ile Asn Ser Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Thr Tyr
325 330 335
Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly Lys Ala
340 345 350
Thr Cys Cys Tyr Ala Tyr Ser Tyr Arg Gly Pro Met Ala Cys Lys Gly
355 360 365
Val Tyr Ser Gly Glu Leu Ser Thr Asn Phe Glu Cys Gly Leu Leu Val
370 375 380
Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Thr Glu Pro
385 390 395 400
Leu Val Leu Thr Gln Tyr Asn Tyr Asn Asn Ile Thr Leu Asp Lys Cys
405 410 415
Val Ala Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Phe Ile Thr Asn
420 425 430
Val Thr Asp Ser Ala Ala Asn Phe Ser Tyr Leu Ala Asp Gly Gly Leu
435 440 445
Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Val Phe Val Val Gln Gly
450 455 460
Ile Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn
465 470 475 480
Gln Gln Phe Val Val Ser Gly Gly Asn Ile Val Gly Ile Leu Thr Ser
485 490 495
Arg Asn Glu Thr Gly Ser Glu Gln Val Glu Asn Gln Phe Tyr Val Lys
500 505 510
Leu Thr Asn Ser Ser His Arg Arg Lys Arg Ser Ile Gly Gln Asn Val
515 520 525
Thr Ser Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys Ile Glu Pro Asp
530 535 540
Gly Ser Leu Lys Met Ile Val Pro Glu Glu Leu Lys Gln Phe Val Ala
545 550 555 560
Pro Leu Leu Asn Ile Thr Glu Ser Val Leu Ile Pro Asn Ser Phe Asn
565 570 575
Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Val Gln
580 585 590
Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Leu Glu Cys Arg Lys
595 600 605
Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val
610 615 620
Asn Ser Val Ser Gln Lys Glu Asp Met Glu Leu Leu Ser Phe Tyr Ser
625 630 635 640
Ser Thr Lys Pro Lys Gly Tyr Asp Thr Pro Val Leu Ser Asn Val Ser
645 650 655
Thr Gly Glu Phe Asn Ile Ser Leu Leu Leu Lys Pro Pro Ser Ser Pro
660 665 670
Ser Gly Arg Ser Phe Ile Glu Glu Leu Leu Phe Thr Ser Val Glu Thr
675 680 685
Val Gly Leu Pro Thr Asp Ala Glu Tyr Lys Lys Cys Thr Ala Gly Pro
690 695 700
Leu Gly Thr Leu Lys Asp Leu Ile Cys Ala Arg Glu Tyr Asn Gly Leu
705 710 715 720
Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr Met Tyr Thr
725 730 735
Ala Ser Leu Val Gly Ala Met Ala Phe Gly Gly Ile Thr Ser Ala Ala
740 745 750
Ala Ile Pro Phe Ala Thr Gln Ile Gln Ala Arg Ile Asn His Leu Gly
755 760 765
Ile Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys Ile Ala Ala Ser
770 775 780
Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr Ser
785 790 795 800
Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile
805 810 815
Leu Thr Glu Thr Met Asn Ser Leu Asn Lys Asn Phe Gly Ala Ile Thr
820 825 830
Ser Val Ile Gln Asp Ile Tyr Ala Gln Leu Asp Ala Ile Gln Ala Asp
835 840 845
Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val
850 855 860
Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile Arg Val Ser Gln Gln Arg
865 870 875 880
Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Asn
885 890 895
Arg Tyr Gly Phe Cys Gly Ser Gly Arg His Val Leu Ser Ile Pro Gln
900 905 910
Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro Glu
915 920 925
Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Asn Pro Ala
930 935 940
Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe
945 950 955 960
Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met
965 970 975
Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln
980 985 990
Ala Asn Tyr Val Asn Val Asn Lys Thr Val Ile Thr Thr Phe Val Glu
995 1000 1005
Asp Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys Trp Trp Asn
1010 1015 1020
Asp Thr Lys His Gln Leu Pro Asp Phe Asp Asp Phe Asn Tyr Thr
1025 1030 1035
Val Pro Ile Leu Asn Ile Ser Gly Glu Ile Asp Tyr Ile Gln Gly
1040 1045 1050
Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Glu Leu
1055 1060 1065
Ser Ile Ile Lys Thr Tyr Ile Lys Trp Pro
1070 1075
<210> 9
<211> 1166
<212> PRT
<213> avian infectious bronchitis Virus
<400> 9
Met Leu Gly Lys Ser Leu Phe Ile Val Thr Leu Leu Phe Ala Leu Cys
1 5 10 15
Ser Ala Ala Leu Phe Asp Asn Asn Glu Thr Val Tyr Tyr Tyr Gln Ser
20 25 30
Ala Phe Arg Pro Ala Asp Gly Trp His Leu His Gly Gly Ala Tyr Ala
35 40 45
Val Val Asn Val Ser Leu Glu Thr Asn Asn Ala Gly Thr Ala Ser Gln
50 55 60
Cys Ile Ala Gly Ala Ile Ser Trp Ser Lys Asn Phe Ser Ala Ser Ala
65 70 75 80
Val Ala Met Thr Ala Pro Glu Leu Gly Met Thr Trp Ser Thr Gly Gln
85 90 95
Phe Cys Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr
100 105 110
His Cys Phe Lys His Gly Asn Gly Leu Cys Pro Leu Thr Gly Leu Ile
115 120 125
Pro Ser Gly Phe Ile Arg Val Ser Ala Met Arg Lys Gly Ser Asn Ser
130 135 140
Leu Phe Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro Arg Phe Lys
145 150 155 160
Ser Leu Gln Cys Val Asn Asn Tyr Thr Ser Val Tyr Leu Asn Gly Asp
165 170 175
Leu Val Phe Thr Ser Asn Glu Thr Lys Pro Val Ser Ala Ala Gly Val
180 185 190
Ser Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Thr Met Ser Glu Val
195 200 205
Lys Val Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Thr Val Ile Pro
210 215 220
Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly
225 230 235 240
Asn Phe Ser Asp Gly Phe Tyr Pro Tyr Thr Asn Ser Ser Leu Val Lys
245 250 255
Glu Arg Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Val
260 265 270
Leu Thr Asn Phe Thr Phe Ser Asn Val Ser Asn Ala Pro Pro Asn Thr
275 280 285
Gly Gly Val His Ser Ile Val Leu His Gln Thr Gln Thr Ala Gln Ser
290 295 300
Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Arg Tyr Val
305 310 315 320
Glu Ser Asp Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Ser Phe Arg
325 330 335
Leu Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser
340 345 350
Leu Gly Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Asn
355 360 365
Asn Met Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Ser Gly Pro Thr Leu
370 375 380
Cys Lys Gly Val Tyr Ser Gly Gln Leu Gln Lys Thr Phe Glu Cys Gly
385 390 395 400
Leu Leu Val Phe Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg
405 410 415
Asn Glu Pro Leu Val Leu Thr Gln His Asn Tyr Asn Asn Ile Thr Leu
420 425 430
Asn Lys Cys Val Glu Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Leu
435 440 445
Ile Thr Asn Ile Thr Asp Ser Ala Ala Asn His Gly Tyr Leu Ala Asp
450 455 460
Gly Gly Leu Ala Val Leu Asp Thr Ser Gly Ala Ile Asp Val Phe Val
465 470 475 480
Val Gln Gly Val Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu
485 490 495
Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Gln Leu Val Gly Ile
500 505 510
Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Pro Ile Glu Asn Arg Phe
515 520 525
Tyr Val Lys Phe Pro Asn Ser Arg Arg Arg Thr Gly Arg Ser Thr Ile
530 535 540
Ala Asn Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile
545 550 555 560
Lys Pro Asp Gly Ser Val Ser Glu Ile Val Pro Gln Glu Ile Glu His
565 570 575
Phe Val Ala Pro Leu Leu Asn Val Thr Glu His Val Leu Ile Pro Asn
580 585 590
Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp
595 600 605
Lys Ile Gln Ile Asn Cys Arg Gln Tyr Val Cys Gly Asn Ser Ile Glu
610 615 620
Cys Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu
625 630 635 640
Ser Val Val Asn Thr Val Gly Gln Arg Glu Asp Met Glu Leu Leu Ser
645 650 655
Phe Tyr Ser Ser Thr Lys Pro Lys Asp Tyr Asn Ile Pro Ile Phe Ser
660 665 670
Asn Val Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Leu Thr Pro Pro
675 680 685
Asn Ser Pro Thr Gly Arg Ser Phe Ile Glu Asp Ile Leu Phe Thr Ser
690 695 700
Val Glu Ser Val Gly Leu Pro Thr Asp Glu Glu Tyr Lys Lys Cys Thr
705 710 715 720
Ala Gly Pro Leu Gly Phe Val Lys Asp Leu Val Cys Ala Arg Glu Tyr
725 730 735
Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr
740 745 750
Met Tyr Thr Ser Thr Leu Val Ala Ser Met Ala Leu Gly Gly Ile Thr
755 760 765
Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn
770 775 780
His Leu Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile
785 790 795 800
Ala Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg
805 810 815
Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln
820 825 830
Ser Ser Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly
835 840 845
Ala Ile Ser Ser Val Leu Gln Asp Ile Tyr Gln Gln Leu Asp Ala Ile
850 855 860
Gln Ala Asp Ala Gln Val Asp Arg Ile Ile Thr Gly Arg Leu Ser Ser
865 870 875 880
Leu Ser Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Tyr Arg Val Ser
885 890 895
Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser
900 905 910
Gln Ser Thr Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr
915 920 925
Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr
930 935 940
Thr Pro Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val
945 950 955 960
Asn Pro Pro Asn Ala Ser Gln Tyr Ala Leu Val Pro Ala Asn Gly Arg
965 970 975
Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp
980 985 990
Met Tyr Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr
995 1000 1005
Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Arg Thr Val Ile Thr
1010 1015 1020
Thr Phe Val Asp Asn Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser
1025 1030 1035
Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Glu
1040 1045 1050
Phe Asn Tyr Thr Ile Pro Val Leu Asn Ile Ser Asn Glu Ile Asp
1055 1060 1065
Arg Ile Gln Glu Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp
1070 1075 1080
Leu Glu Ala Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp
1085 1090 1095
Tyr Val Trp Leu Ala Ile Ala Phe Leu Thr Ile Ile Phe Ile Leu
1100 1105 1110
Val Leu Cys Trp Ile Phe Phe Met Thr Gly Cys Cys Gly Cys Cys
1115 1120 1125
Cys Gly Cys Phe Gly Ile Met Pro Leu Met Ser Lys Cys Gly Lys
1130 1135 1140
Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val Thr Glu
1145 1150 1155
Gln Tyr Arg Pro Lys Lys Ser Val
1160 1165
<210> 10
<211> 1079
<212> PRT
<213> avian infectious bronchitis Virus
<400> 10
Ala Leu Phe Asp Asn Asn Glu Thr Val Tyr Tyr Tyr Gln Ser Ala Phe
1 5 10 15
Arg Pro Ala Asp Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val
20 25 30
Asn Val Ser Leu Glu Thr Asn Asn Ala Gly Thr Ala Ser Gln Cys Ile
35 40 45
Ala Gly Ala Ile Ser Trp Ser Lys Asn Phe Ser Ala Ser Ala Val Ala
50 55 60
Met Thr Ala Pro Glu Leu Gly Met Thr Trp Ser Thr Gly Gln Phe Cys
65 70 75 80
Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr His Cys
85 90 95
Phe Lys His Gly Asn Gly Leu Cys Pro Leu Thr Gly Leu Ile Pro Ser
100 105 110
Gly Phe Ile Arg Val Ser Ala Met Arg Lys Gly Ser Asn Ser Leu Phe
115 120 125
Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro Arg Phe Lys Ser Leu
130 135 140
Gln Cys Val Asn Asn Tyr Thr Ser Val Tyr Leu Asn Gly Asp Leu Val
145 150 155 160
Phe Thr Ser Asn Glu Thr Lys Pro Val Ser Ala Ala Gly Val Ser Phe
165 170 175
Lys Ala Gly Gly Pro Ile Thr Tyr Lys Thr Met Ser Glu Val Lys Val
180 185 190
Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Thr Val Ile Pro Cys Asp
195 200 205
Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe
210 215 220
Ser Asp Gly Phe Tyr Pro Tyr Thr Asn Ser Ser Leu Val Lys Glu Arg
225 230 235 240
Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Val Leu Thr
245 250 255
Asn Phe Thr Phe Ser Asn Val Ser Asn Ala Pro Pro Asn Thr Gly Gly
260 265 270
Val His Ser Ile Val Leu His Gln Thr Gln Thr Ala Gln Ser Gly Tyr
275 280 285
Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Arg Tyr Val Glu Ser
290 295 300
Asp Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Ser Phe Arg Leu Glu
305 310 315 320
Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Gly
325 330 335
Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Asn Asn Met
340 345 350
Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Ser Gly Pro Thr Leu Cys Lys
355 360 365
Gly Val Tyr Ser Gly Gln Leu Gln Lys Thr Phe Glu Cys Gly Leu Leu
370 375 380
Val Phe Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Asn Glu
385 390 395 400
Pro Leu Val Leu Thr Gln His Asn Tyr Asn Asn Ile Thr Leu Asn Lys
405 410 415
Cys Val Glu Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly Leu Ile Thr
420 425 430
Asn Ile Thr Asp Ser Ala Ala Asn His Gly Tyr Leu Ala Asp Gly Gly
435 440 445
Leu Ala Val Leu Asp Thr Ser Gly Ala Ile Asp Val Phe Val Val Gln
450 455 460
Gly Val Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val
465 470 475 480
Asn Gln Gln Phe Val Val Ser Gly Gly Gln Leu Val Gly Ile Leu Thr
485 490 495
Ser Arg Asn Glu Thr Gly Ser Gln Pro Ile Glu Asn Arg Phe Tyr Val
500 505 510
Lys Phe Pro Asn Ser Arg Arg Arg Thr Gly Arg Ser Thr Ile Ala Asn
515 520 525
Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro
530 535 540
Asp Gly Ser Val Ser Glu Ile Val Pro Gln Glu Ile Glu His Phe Val
545 550 555 560
Ala Pro Leu Leu Asn Val Thr Glu His Val Leu Ile Pro Asn Ser Phe
565 570 575
Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Asp Lys Ile
580 585 590
Gln Ile Asn Cys Arg Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys Arg
595 600 605
Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val
610 615 620
Val Asn Thr Val Gly Gln Arg Glu Asp Met Glu Leu Leu Ser Phe Tyr
625 630 635 640
Ser Ser Thr Lys Pro Lys Asp Tyr Asn Ile Pro Ile Phe Ser Asn Val
645 650 655
Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Leu Thr Pro Pro Asn Ser
660 665 670
Pro Thr Gly Arg Ser Phe Ile Glu Asp Ile Leu Phe Thr Ser Val Glu
675 680 685
Ser Val Gly Leu Pro Thr Asp Glu Glu Tyr Lys Lys Cys Thr Ala Gly
690 695 700
Pro Leu Gly Phe Val Lys Asp Leu Val Cys Ala Arg Glu Tyr Asn Gly
705 710 715 720
Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr Met Tyr
725 730 735
Thr Ser Thr Leu Val Ala Ser Met Ala Leu Gly Gly Ile Thr Ala Ala
740 745 750
Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu
755 760 765
Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala
770 775 780
Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr
785 790 795 800
Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ser
805 810 815
Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile
820 825 830
Ser Ser Val Leu Gln Asp Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala
835 840 845
Asp Ala Gln Val Asp Arg Ile Ile Thr Gly Arg Leu Ser Ser Leu Ser
850 855 860
Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Tyr Arg Val Ser Gln Gln
865 870 875 880
Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser
885 890 895
Thr Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro
900 905 910
Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro
915 920 925
Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Asn Pro
930 935 940
Pro Asn Ala Ser Gln Tyr Ala Leu Val Pro Ala Asn Gly Arg Gly Ile
945 950 955 960
Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr
965 970 975
Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys
980 985 990
Gln Ala Asn Tyr Val Ser Val Asn Arg Thr Val Ile Thr Thr Phe Val
995 1000 1005
Asp Asn Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys Trp Trp
1010 1015 1020
Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Glu Phe Asn Tyr
1025 1030 1035
Thr Ile Pro Val Leu Asn Ile Ser Asn Glu Ile Asp Arg Ile Gln
1040 1045 1050
Glu Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Ala
1055 1060 1065
Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro
1070 1075
<210> 11
<211> 1159
<212> PRT
<213> avian infectious bronchitis Virus
<400> 11
Met Leu Val Lys Ser Leu Phe Leu Val Thr Ile Leu Phe Ala Leu Cys
1 5 10 15
Ser Ala Asn Leu Tyr Asp Asn Glu Ser Phe Val Tyr Tyr Tyr Gln Ser
20 25 30
Ala Phe Arg Pro Gly His Gly Trp His Leu His Gly Gly Ala Tyr Ala
35 40 45
Val Val Asn Val Ser Ser Glu Asn Asn Asn Ala Gly Thr Ala Pro Ser
50 55 60
Cys Thr Ala Gly Ala Ile Gly Tyr Ser Lys Asn Phe Ser Ala Ala Ser
65 70 75 80
Val Ala Met Thr Ala Pro Leu Ser Gly Met Ser Trp Ser Ala Ser Ser
85 90 95
Phe Cys Thr Ala His Cys Asn Phe Thr Ser Tyr Ile Val Phe Val Thr
100 105 110
His Cys Phe Lys Ser Gly Ser Asn Ser Cys Pro Leu Thr Gly Leu Ile
115 120 125
Pro Ser Gly Tyr Ile Arg Ile Ala Ala Met Lys His Gly Ser Arg Thr
130 135 140
Pro Gly His Leu Phe Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro
145 150 155 160
Lys Phe Arg Ser Leu Gln Cys Val Asn Asn His Thr Ser Val Tyr Leu
165 170 175
Asn Gly Asp Leu Val Phe Thr Ser Gly Tyr Thr Glu Asp Val Val Ala
180 185 190
Ala Gly Val His Phe Lys Ser Gly Gly Pro Ile Thr Tyr Lys Val Met
195 200 205
Arg Glu Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp
210 215 220
Val Ile Leu Cys Asp Asp Thr Pro Arg Gly Leu Leu Ala Cys Gln Tyr
225 230 235 240
Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Thr Ser
245 250 255
Ile Val Lys Asp Lys Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr
260 265 270
Thr Leu Thr Leu Thr Asn Phe Thr Phe Ser Asn Glu Ser Gly Ala Pro
275 280 285
Pro Asn Thr Gly Gly Val Asp Ser Phe Ile Leu Tyr Gln Thr Gln Thr
290 295 300
Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe
305 310 315 320
Val Tyr Arg Glu Ser Asn Tyr Met Tyr Gly Ser Tyr His Pro Arg Cys
325 330 335
Ser Phe Arg Pro Glu Thr Leu Asn Gly Leu Trp Phe Asn Ser Leu Ser
340 345 350
Val Ser Leu Thr Tyr Gly Pro Ile Gln Gly Gly Cys Lys Gln Ser Val
355 360 365
Phe Asn Gly Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro
370 375 380
Arg Ala Cys Lys Gly Val Tyr Arg Gly Glu Leu Thr Gln His Phe Glu
385 390 395 400
Cys Gly Leu Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln
405 410 415
Thr Ala Thr Gln Pro Pro Val Leu Thr Gln Asn Phe Tyr Asn Asn Ile
420 425 430
Thr Leu Gly Lys Cys Val Asp Tyr Asn Val Tyr Gly Arg Thr Gly Gln
435 440 445
Gly Phe Ile Thr Asn Val Thr Asp Leu Ala Thr Ser His Asn Tyr Leu
450 455 460
Ala Asp Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile
465 470 475 480
Phe Val Val Gln Gly Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Leu
485 490 495
Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val
500 505 510
Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Pro Leu Glu Asn
515 520 525
Gln Phe Tyr Ile Lys Ile Thr Asn Gly Thr His Arg Ser Arg Arg Ser
530 535 540
Val Asn Glu Asn Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe
545 550 555 560
Cys Ile Lys Pro Asp Gly Ser Val Ser Pro Ile Val Pro Lys Glu Leu
565 570 575
Glu Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile
580 585 590
Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr His
595 600 605
Met Asp Lys Ile Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser
610 615 620
Leu Ala Cys Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn
625 630 635 640
Ile Leu Ser Val Val Asn Ser Val Gly Gln Lys Glu Asp Met Glu Leu
645 650 655
Leu Ser Phe Tyr Ser Ser Thr Lys Pro Ser Gly Phe Asn Thr Pro Val
660 665 670
Phe Ser Asn Leu Ser Thr Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr
675 680 685
Thr Pro Ser Ser Pro Arg Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe
690 695 700
Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp Glu Ala Tyr Lys Lys
705 710 715 720
Cys Thr Ala Gly Pro Leu Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg
725 730 735
Glu Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met
740 745 750
Gln Thr Leu Tyr Thr Ser Ser Leu Val Val Ser Met Ala Phe Gly Gly
755 760 765
Ile Thr Ser Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg
770 775 780
Ile Asn His Leu Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu
785 790 795 800
Lys Ile Ala Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly
805 810 815
Phe Arg Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn
820 825 830
Lys Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn
835 840 845
Phe Gly Ala Ile Ser Ser Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp
850 855 860
Ala Ile Gln Ala Asn Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu
865 870 875 880
Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ala Glu Tyr Ile Arg
885 890 895
Val Ser Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val
900 905 910
Lys Ser Gln Ser Ile Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val
915 920 925
Leu Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe
930 935 940
Ser Tyr Thr Pro Asp Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe
945 950 955 960
Cys Val Lys Pro Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn
965 970 975
Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala
980 985 990
Arg Asp Met Tyr Met Pro Arg Ala Ile Thr Ala Gly Asp Ile Val Thr
995 1000 1005
Leu Thr Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Lys Thr Val
1010 1015 1020
Ile Thr Thr Phe Val Asp Asn Asp Asp Phe Asp Phe Asn Asp Glu
1025 1030 1035
Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe
1040 1045 1050
Asp Lys Phe Asn Tyr Thr Val Pro Ile Leu Asp Ile Asp Ser Glu
1055 1060 1065
Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu
1070 1075 1080
Ile Asp Leu Glu Lys Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp
1085 1090 1095
Pro Trp Tyr Val Trp Leu Ala Ile Ala Phe Ala Thr Ile Ile Phe
1100 1105 1110
Ile Leu Ile Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly
1115 1120 1125
Cys Cys Cys Gly Cys Phe Gly Ile Met Pro Leu Met Ser Lys Cys
1130 1135 1140
Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val
1145 1150 1155
Thr
<210> 12
<211> 1081
<212> PRT
<213> avian infectious bronchitis Virus
<400> 12
Asn Leu Tyr Asp Asn Glu Ser Phe Val Tyr Tyr Tyr Gln Ser Ala Phe
1 5 10 15
Arg Pro Gly His Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val
20 25 30
Asn Val Ser Ser Glu Asn Asn Asn Ala Gly Thr Ala Pro Ser Cys Thr
35 40 45
Ala Gly Ala Ile Gly Tyr Ser Lys Asn Phe Ser Ala Ala Ser Val Ala
50 55 60
Met Thr Ala Pro Leu Ser Gly Met Ser Trp Ser Ala Ser Ser Phe Cys
65 70 75 80
Thr Ala His Cys Asn Phe Thr Ser Tyr Ile Val Phe Val Thr His Cys
85 90 95
Phe Lys Ser Gly Ser Asn Ser Cys Pro Leu Thr Gly Leu Ile Pro Ser
100 105 110
Gly Tyr Ile Arg Ile Ala Ala Met Lys His Gly Ser Arg Thr Pro Gly
115 120 125
His Leu Phe Tyr Asn Leu Thr Val Ser Val Thr Lys Tyr Pro Lys Phe
130 135 140
Arg Ser Leu Gln Cys Val Asn Asn His Thr Ser Val Tyr Leu Asn Gly
145 150 155 160
Asp Leu Val Phe Thr Ser Gly Tyr Thr Glu Asp Val Val Ala Ala Gly
165 170 175
Val His Phe Lys Ser Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu
180 185 190
Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp Val Ile
195 200 205
Leu Cys Asp Asp Thr Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr
210 215 220
Gly Asn Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Thr Ser Ile Val
225 230 235 240
Lys Asp Lys Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu
245 250 255
Thr Leu Thr Asn Phe Thr Phe Ser Asn Glu Ser Gly Ala Pro Pro Asn
260 265 270
Thr Gly Gly Val Asp Ser Phe Ile Leu Tyr Gln Thr Gln Thr Ala Gln
275 280 285
Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr
290 295 300
Arg Glu Ser Asn Tyr Met Tyr Gly Ser Tyr His Pro Arg Cys Ser Phe
305 310 315 320
Arg Pro Glu Thr Leu Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser
325 330 335
Leu Thr Tyr Gly Pro Ile Gln Gly Gly Cys Lys Gln Ser Val Phe Asn
340 345 350
Gly Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Arg Ala
355 360 365
Cys Lys Gly Val Tyr Arg Gly Glu Leu Thr Gln His Phe Glu Cys Gly
370 375 380
Leu Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Ala
385 390 395 400
Thr Gln Pro Pro Val Leu Thr Gln Asn Phe Tyr Asn Asn Ile Thr Leu
405 410 415
Gly Lys Cys Val Asp Tyr Asn Val Tyr Gly Arg Thr Gly Gln Gly Phe
420 425 430
Ile Thr Asn Val Thr Asp Leu Ala Thr Ser His Asn Tyr Leu Ala Asp
435 440 445
Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val
450 455 460
Val Gln Gly Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Leu Cys Glu
465 470 475 480
Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile
485 490 495
Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Pro Leu Glu Asn Gln Phe
500 505 510
Tyr Ile Lys Ile Thr Asn Gly Thr His Arg Ser Arg Arg Ser Val Asn
515 520 525
Glu Asn Val Thr Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile
530 535 540
Lys Pro Asp Gly Ser Val Ser Pro Ile Val Pro Lys Glu Leu Glu Gln
545 550 555 560
Phe Val Ala Pro Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn
565 570 575
Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr His Met Asp
580 585 590
Lys Ile Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Leu Ala
595 600 605
Cys Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu
610 615 620
Ser Val Val Asn Ser Val Gly Gln Lys Glu Asp Met Glu Leu Leu Ser
625 630 635 640
Phe Tyr Ser Ser Thr Lys Pro Ser Gly Phe Asn Thr Pro Val Phe Ser
645 650 655
Asn Leu Ser Thr Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr Thr Pro
660 665 670
Ser Ser Pro Arg Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser
675 680 685
Val Glu Ser Val Gly Leu Pro Thr Asp Glu Ala Tyr Lys Lys Cys Thr
690 695 700
Ala Gly Pro Leu Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr
705 710 715 720
Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr
725 730 735
Leu Tyr Thr Ser Ser Leu Val Val Ser Met Ala Phe Gly Gly Ile Thr
740 745 750
Ser Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn
755 760 765
His Leu Gly Ile Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile
770 775 780
Ala Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg
785 790 795 800
Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln
805 810 815
Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly
820 825 830
Ala Ile Ser Ser Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile
835 840 845
Gln Ala Asn Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser
850 855 860
Leu Ser Val Leu Ala Ser Ala Lys Gln Ala Glu Tyr Ile Arg Val Ser
865 870 875 880
Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser
885 890 895
Gln Ser Ile Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr
900 905 910
Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr
915 920 925
Thr Pro Asp Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val
930 935 940
Lys Pro Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg
945 950 955 960
Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp
965 970 975
Met Tyr Met Pro Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr
980 985 990
Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr
995 1000 1005
Phe Val Asp Asn Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys
1010 1015 1020
Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Lys Phe
1025 1030 1035
Asn Tyr Thr Val Pro Ile Leu Asp Ile Asp Ser Glu Ile Asp Arg
1040 1045 1050
Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu
1055 1060 1065
Glu Lys Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro
1070 1075 1080
<210> 13
<211> 1156
<212> PRT
<213> avian infectious bronchitis Virus
<400> 13
Met Leu Val Lys Ser Leu Phe Ile Val Thr Leu Leu Phe Ala Leu Cys
1 5 10 15
Ser Ala Ala Leu Phe Asp Asn Asn Gln Ala Val Tyr Tyr Tyr Gln Ser
20 25 30
Ala Phe Arg Pro Ser Ser Gly Trp His Lys His Gly Gly Ala Tyr Ala
35 40 45
Val Ala Asn Val Ser Leu Glu Tyr Ala Asn Ala Gly Ser Ser Thr His
50 55 60
Cys Thr Ala Gly Ala Ile Tyr Trp Ser Lys Asn Phe Thr Ala Ser Ser
65 70 75 80
Val Ala Met Thr Ala Pro Gly Thr Gly Met Ser Trp Ser Thr Ala Gln
85 90 95
Phe Cys Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr
100 105 110
His Cys Tyr Lys Ser Gly Asp Val Cys Pro Leu Thr Gly Leu Ile Pro
115 120 125
Ser Gly Tyr Ile Arg Ile Ser Ala Met Thr Lys Gly Thr Thr Ser Leu
130 135 140
Phe Tyr Asn Leu Thr Val Pro Val Thr Lys Tyr Pro Lys Phe Lys Ser
145 150 155 160
Leu Gln Cys Val Asp Asn Phe Thr Ser Val Tyr Leu Asn Gly Asp Leu
165 170 175
Val Phe Thr Ser Asn Glu Thr Lys Asp Val Ser Ala Ala Gly Val His
180 185 190
Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Glu Lys Val Asp
195 200 205
Val Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys
210 215 220
Asp Asn Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn
225 230 235 240
Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Ile Ser Leu Val Lys Glu
245 250 255
Lys Phe Ile Val Tyr Arg Glu Thr Ser Val Asn Thr Thr Leu Val Leu
260 265 270
Thr Asn Phe Thr Phe Thr Asn Val Ser Asn Ala Leu Pro Asn Thr Gly
275 280 285
Gly Val Asn Thr Ile Asn Ile Tyr Gln Thr Gln Thr Ala Gln Ser Gly
290 295 300
Cys Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Gln
305 310 315 320
Ser Asp Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Asp Phe Arg Pro
325 330 335
Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu
340 345 350
Ala Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Asn
355 360 365
Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly Pro Arg Leu Cys
370 375 380
Lys Gly Val Tyr Ile Gly Glu Leu Gln Gln Tyr Phe Glu Cys Gly Leu
385 390 395 400
Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Asn
405 410 415
Glu Pro Leu Val Leu Thr His His Asn Tyr Asn Asn Ile Thr Leu Asp
420 425 430
Arg Cys Val Glu Tyr Asn Ile Tyr Gly Arg Ser Gly Gln Gly Phe Ile
435 440 445
Thr Asn Val Thr Ala Ala Ala Ala Asn Tyr Asn Tyr Leu Ala Asp Gly
450 455 460
Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val
465 470 475 480
Gln Gly Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp
485 490 495
Val Asn Gln Gln Phe Val Val Ser Gly Gly Gly Ile Val Gly Val Leu
500 505 510
Thr Ser His Asn Glu Thr Gly Ser Gln Gln Leu Glu Asn Leu Phe Tyr
515 520 525
Val Lys Leu Thr Asn Ser Thr Arg Arg Thr Arg Arg Ser Thr Ile Ala
530 535 540
Asn Val Thr Thr Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys Ile Lys
545 550 555 560
Pro Asp Gly Leu Val Ser Glu Ile Val Pro Gln Glu Leu Asp Tyr Phe
565 570 575
Val Ala Pro Leu Leu Asn Val Thr Glu His Val Leu Ile Pro Asn Ser
580 585 590
Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Glu Lys
595 600 605
Val Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys
610 615 620
Arg Asn Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser
625 630 635 640
Ile Val Asn Ser Val Gly Gln Arg Glu Asp Met Glu Ser Leu Thr Phe
645 650 655
Tyr Ser Ser Thr Lys Pro Lys Gly Tyr Asn Thr Pro Ile Phe Ser Asn
660 665 670
Ile Ser Thr Gly Asp Phe Asn Ile Ser Leu Met Leu Thr Pro Pro Ser
675 680 685
Ser Pro Ser Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val
690 695 700
Glu Thr Val Gly Leu Pro Thr Asp Ala Glu Tyr Lys Lys Cys Thr Ala
705 710 715 720
Gly Pro Leu Gly Thr Leu Lys Asp Leu Ile Cys Ala Arg Glu Tyr Asn
725 730 735
Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr Met
740 745 750
Tyr Thr Ala Ser Leu Val Gly Ala Met Ala Phe Gly Gly Ile Thr Ser
755 760 765
Ala Ala Ala Ile Pro Phe Ala Thr Gln Ile Gln Ala Arg Ile Asn His
770 775 780
Leu Gly Ile Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys Ile Ala
785 790 795 800
Ala Ser Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser
805 810 815
Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser
820 825 830
Ala Ile Leu Thr Glu Thr Met Asn Ser Leu Asn Lys Asn Phe Gly Ala
835 840 845
Ile Thr Ser Val Ile Gln Asp Ile Tyr Ala Gln Leu Asp Ala Ile Gln
850 855 860
Ala Asp Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu
865 870 875 880
Ser Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile Arg Val Ser Gln
885 890 895
Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln
900 905 910
Ser Asn Arg Tyr Gly Phe Cys Gly Ser Gly Arg His Val Leu Ser Ile
915 920 925
Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr
930 935 940
Pro Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Ser
945 950 955 960
Pro Ala Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly
965 970 975
Ile Phe Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr Ala Arg Asp Met
980 985 990
Tyr Met Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser
995 1000 1005
Cys Gln Ala Asn Tyr Val Asn Val Asn Lys Thr Val Ile Thr Thr
1010 1015 1020
Phe Val Glu Asp Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys
1025 1030 1035
Trp Trp Asn Glu Thr Lys His Glu Ile Pro Asp Phe Asp Glu Phe
1040 1045 1050
Asn Tyr Thr Val Pro Ile Leu Asn Ile Ser Ser Glu Ile Asp Arg
1055 1060 1065
Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asn Leu
1070 1075 1080
Glu Glu Leu Ser Ile Ile Lys Thr Tyr Ile Lys Trp Pro Trp Tyr
1085 1090 1095
Val Trp Leu Ala Ile Gly Phe Ala Ile Ile Ile Phe Ile Leu Ile
1100 1105 1110
Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys
1115 1120 1125
Gly Cys Phe Gly Ile Ile Pro Leu Met Ser Lys Cys Gly Lys Lys
1130 1135 1140
Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val Val Thr
1145 1150 1155
<210> 14
<211> 1078
<212> PRT
<213> avian infectious bronchitis Virus
<400> 14
Ala Leu Phe Asp Asn Asn Gln Ala Val Tyr Tyr Tyr Gln Ser Ala Phe
1 5 10 15
Arg Pro Ser Ser Gly Trp His Lys His Gly Gly Ala Tyr Ala Val Ala
20 25 30
Asn Val Ser Leu Glu Tyr Ala Asn Ala Gly Ser Ser Thr His Cys Thr
35 40 45
Ala Gly Ala Ile Tyr Trp Ser Lys Asn Phe Thr Ala Ser Ser Val Ala
50 55 60
Met Thr Ala Pro Gly Thr Gly Met Ser Trp Ser Thr Ala Gln Phe Cys
65 70 75 80
Thr Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr His Cys
85 90 95
Tyr Lys Ser Gly Asp Val Cys Pro Leu Thr Gly Leu Ile Pro Ser Gly
100 105 110
Tyr Ile Arg Ile Ser Ala Met Thr Lys Gly Thr Thr Ser Leu Phe Tyr
115 120 125
Asn Leu Thr Val Pro Val Thr Lys Tyr Pro Lys Phe Lys Ser Leu Gln
130 135 140
Cys Val Asp Asn Phe Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe
145 150 155 160
Thr Ser Asn Glu Thr Lys Asp Val Ser Ala Ala Gly Val His Phe Lys
165 170 175
Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Glu Lys Val Asp Val Leu
180 185 190
Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys Asp Asn
195 200 205
Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser
210 215 220
Asp Gly Phe Tyr Pro Phe Thr Asn Ile Ser Leu Val Lys Glu Lys Phe
225 230 235 240
Ile Val Tyr Arg Glu Thr Ser Val Asn Thr Thr Leu Val Leu Thr Asn
245 250 255
Phe Thr Phe Thr Asn Val Ser Asn Ala Leu Pro Asn Thr Gly Gly Val
260 265 270
Asn Thr Ile Asn Ile Tyr Gln Thr Gln Thr Ala Gln Ser Gly Cys Tyr
275 280 285
Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Gln Ser Asp
290 295 300
Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Asp Phe Arg Pro Glu Thr
305 310 315 320
Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Ala Tyr
325 330 335
Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Asn Arg Ala
340 345 350
Thr Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly Pro Arg Leu Cys Lys Gly
355 360 365
Val Tyr Ile Gly Glu Leu Gln Gln Tyr Phe Glu Cys Gly Leu Leu Val
370 375 380
Tyr Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Asn Glu Pro
385 390 395 400
Leu Val Leu Thr His His Asn Tyr Asn Asn Ile Thr Leu Asp Arg Cys
405 410 415
Val Glu Tyr Asn Ile Tyr Gly Arg Ser Gly Gln Gly Phe Ile Thr Asn
420 425 430
Val Thr Ala Ala Ala Ala Asn Tyr Asn Tyr Leu Ala Asp Gly Gly Leu
435 440 445
Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln Gly
450 455 460
Glu Tyr Gly Pro Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn
465 470 475 480
Gln Gln Phe Val Val Ser Gly Gly Gly Ile Val Gly Val Leu Thr Ser
485 490 495
His Asn Glu Thr Gly Ser Gln Gln Leu Glu Asn Leu Phe Tyr Val Lys
500 505 510
Leu Thr Asn Ser Thr Arg Arg Thr Arg Arg Ser Thr Ile Ala Asn Val
515 520 525
Thr Thr Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys Ile Lys Pro Asp
530 535 540
Gly Leu Val Ser Glu Ile Val Pro Gln Glu Leu Asp Tyr Phe Val Ala
545 550 555 560
Pro Leu Leu Asn Val Thr Glu His Val Leu Ile Pro Asn Ser Phe Asn
565 570 575
Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met Glu Lys Val Gln
580 585 590
Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Ile Glu Cys Arg Asn
595 600 605
Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Ile Val
610 615 620
Asn Ser Val Gly Gln Arg Glu Asp Met Glu Ser Leu Thr Phe Tyr Ser
625 630 635 640
Ser Thr Lys Pro Lys Gly Tyr Asn Thr Pro Ile Phe Ser Asn Ile Ser
645 650 655
Thr Gly Asp Phe Asn Ile Ser Leu Met Leu Thr Pro Pro Ser Ser Pro
660 665 670
Ser Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Thr
675 680 685
Val Gly Leu Pro Thr Asp Ala Glu Tyr Lys Lys Cys Thr Ala Gly Pro
690 695 700
Leu Gly Thr Leu Lys Asp Leu Ile Cys Ala Arg Glu Tyr Asn Gly Leu
705 710 715 720
Leu Val Leu Pro Pro Ile Ile Thr Ala Asp Met Gln Thr Met Tyr Thr
725 730 735
Ala Ser Leu Val Gly Ala Met Ala Phe Gly Gly Ile Thr Ser Ala Ala
740 745 750
Ala Ile Pro Phe Ala Thr Gln Ile Gln Ala Arg Ile Asn His Leu Gly
755 760 765
Ile Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys Ile Ala Ala Ser
770 775 780
Phe Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr Ser
785 790 795 800
Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile
805 810 815
Leu Thr Glu Thr Met Asn Ser Leu Asn Lys Asn Phe Gly Ala Ile Thr
820 825 830
Ser Val Ile Gln Asp Ile Tyr Ala Gln Leu Asp Ala Ile Gln Ala Asp
835 840 845
Ala Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val
850 855 860
Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile Arg Val Ser Gln Gln Arg
865 870 875 880
Glu Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Asn
885 890 895
Arg Tyr Gly Phe Cys Gly Ser Gly Arg His Val Leu Ser Ile Pro Gln
900 905 910
Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr Tyr Thr Pro Glu
915 920 925
Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Ser Pro Ala
930 935 940
Asn Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe
945 950 955 960
Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met
965 970 975
Pro Arg Asp Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln
980 985 990
Ala Asn Tyr Val Asn Val Asn Lys Thr Val Ile Thr Thr Phe Val Glu
995 1000 1005
Asp Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys Trp Trp Asn
1010 1015 1020
Glu Thr Lys His Glu Ile Pro Asp Phe Asp Glu Phe Asn Tyr Thr
1025 1030 1035
Val Pro Ile Leu Asn Ile Ser Ser Glu Ile Asp Arg Ile Gln Gly
1040 1045 1050
Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asn Leu Glu Glu Leu
1055 1060 1065
Ser Ile Ile Lys Thr Tyr Ile Lys Trp Pro
1070 1075
<210> 15
<211> 1169
<212> PRT
<213> avian infectious bronchitis Virus
<400> 15
Met Leu Val Gln Pro Leu Leu Leu Val Thr Leu Leu Cys Ala Leu Cys
1 5 10 15
Ser Ala Ser Leu Tyr Asn Asn Asp Ser Tyr Val Tyr Tyr Tyr Gln Ser
20 25 30
Ala Phe Arg Pro Phe Asn Gly Trp His Leu His Gly Gly Ala Tyr Ala
35 40 45
Val Val Asn Val Ser Gln Glu Thr Ala Asn Ala Gly Ser Ser Pro Ser
50 55 60
Cys Thr Ala Gly Ala Ile Tyr Trp Ser Lys Asn Phe Thr Ala Ser Ser
65 70 75 80
Val Ala Met Thr Ala Pro Leu Gln Gly Met Gln Trp Ser Thr Ile Gln
85 90 95
Phe Cys Thr Ala His Cys Asn Phe Thr Asn Ile Val Val Phe Val Thr
100 105 110
His Cys Tyr Lys Ser Gly Ser Thr Val Cys Pro Leu Thr Gly Leu Ile
115 120 125
Pro Gln Asn His Ile Arg Ile Ser Ala Met Lys Gln Gly Asn Asn Gly
130 135 140
Pro Ser Gly Leu Phe Tyr Asn Leu Thr Val Pro Val Thr Lys Tyr Ser
145 150 155 160
Lys Phe Lys Ser Leu Gln Cys Val Asn Asn Gln Thr Ser Val Tyr Leu
165 170 175
Asn Gly Asp Leu Val Phe Thr Ser Asn Glu Thr Lys Asp Val Ser Gly
180 185 190
Ala Gly Val Tyr Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met
195 200 205
Arg Glu Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp
210 215 220
Val Ile Leu Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr
225 230 235 240
Asn Thr Gly Lys Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Asp Thr
245 250 255
Leu Val Lys Glu Lys Phe Ile Val Tyr Arg Glu Asn Ser Val Asn Thr
260 265 270
Thr Leu Thr Leu Thr Asn Phe Thr Phe Tyr Asn Glu Ser Asn Ala Leu
275 280 285
Pro Asn Asn Gly Gly Val Asp Thr Ile Gln Leu Tyr Gln Thr His Thr
290 295 300
Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe
305 310 315 320
Gln Tyr Val Glu Ser Asn Phe Met Tyr Gly Ser Tyr His Pro Lys Cys
325 330 335
Gly Phe Arg Pro Glu Ser Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu
340 345 350
Ser Val Ser Leu Ala Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser
355 360 365
Val Phe His Gly Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly
370 375 380
Pro Thr Leu Cys Lys Gly Val Tyr Ser Gly Glu Leu Thr Arg Ser Tyr
385 390 395 400
Gln Cys Gly Leu Leu Val Phe Val Thr Lys Ser Asp Gly Ser Arg Ile
405 410 415
Gln Thr Ser Thr Lys Pro Ile Val Leu Thr Gln His Asn Tyr Asn Asn
420 425 430
Ile Thr Leu Asp Arg Cys Val Glu Tyr Asn Ile Tyr Gly Arg Val Gly
435 440 445
Gln Gly Phe Ile Thr Asn Val Thr Glu Ser Ala Ala Ala Phe Asn Tyr
450 455 460
Leu Glu Asp Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp
465 470 475 480
Ile Phe Val Val Gln Gly Glu Tyr Gly Phe Asn Tyr Tyr Lys Val Asn
485 490 495
Pro Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Asn Leu
500 505 510
Val Gly Ile Leu Thr Ser Ile Asn Gln Thr Gly Ser Gln Ser Ile Glu
515 520 525
Asn Gln Phe Tyr Val Lys Leu Thr Asn Gly Ser Arg Arg Ser Arg Arg
530 535 540
Ser Val Ser Glu Asn Val Thr Ser Cys Pro Tyr Val Ser Tyr Gly Lys
545 550 555 560
Phe Cys Ile Lys Pro Asp Gly Ser Leu Ser Thr Ile Val Pro Lys Glu
565 570 575
Leu Glu Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu His Val Leu
580 585 590
Ile Pro Asp Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr
595 600 605
Arg Met Asp Lys Val Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn
610 615 620
Ser Phe Glu Cys Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp
625 630 635 640
Asn Ile Leu Ser Val Val Asn Ser Val Gly Gln Lys Glu Asp Met Glu
645 650 655
Leu Leu Ser Phe Tyr Ser Ser Thr Lys Pro Ser Gly Ile Ser Gln Pro
660 665 670
Leu Phe Asn Asn Phe Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Leu
675 680 685
Thr Ser Pro Ser Ser Pro Ser Gly Arg Ser Phe Ile Glu Asp Leu Leu
690 695 700
Phe Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp Glu Ala Tyr Lys
705 710 715 720
Lys Cys Thr Ser Gly Pro Leu Gly Phe Val Lys Asp Leu Val Cys Ala
725 730 735
Arg Glu Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu
740 745 750
Met Gln Thr Met Tyr Thr Ser Ser Leu Val Ala Ser Met Ala Leu Gly
755 760 765
Gly Ile Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala
770 775 780
Arg Ile Asn His Leu Gly Ile Thr Gln Ser Leu Leu Met Lys Asn Gln
785 790 795 800
Glu Lys Ile Ala Ala Ser Phe Asn Arg Ala Ile Gly His Met Gln Glu
805 810 815
Gly Phe Lys Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val
820 825 830
Asn Lys Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys
835 840 845
Asn Phe Gly Ala Ile Ser Ser Val Leu Gln Asp Ile Tyr Gln Gln Leu
850 855 860
Asp Val Ile Gln Ala Asp Ala Gln Val Asp Arg Ile Ile Thr Gly Arg
865 870 875 880
Leu Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile
885 890 895
Ala Val Ser Gln Gln Arg Ala Leu Ala Thr Gln Lys Ile Asn Glu Cys
900 905 910
Val Lys Ser Gln Ser Thr Arg Tyr Ser Phe Cys Gly Asn Gly Arg His
915 920 925
Val Leu Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His
930 935 940
Phe Thr Tyr Thr Pro Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly
945 950 955 960
Phe Cys Val Lys Pro Pro Asn Ala Ser His Tyr Ala Ile Val Pro Val
965 970 975
Asn Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr
980 985 990
Ser Arg Asp Met Tyr Met Pro Arg Asn Ile Thr Ala Gly Asp Ile Val
995 1000 1005
Thr Leu Thr Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Lys Thr
1010 1015 1020
Val Ile Ser Thr Phe Val Glu Asp Asp Asp Phe Asp Phe Asp Asp
1025 1030 1035
Glu Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp
1040 1045 1050
Phe Asp Glu Phe Asn Tyr Thr Ile Pro Val Leu Asn Ile Ser Asn
1055 1060 1065
Glu Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser
1070 1075 1080
Leu Ile Asp Leu Glu Thr Leu Ser Ile Leu Lys Thr Tyr Ile Lys
1085 1090 1095
Trp Pro Trp Tyr Val Trp Leu Ala Ile Phe Phe Ala Ile Val Ile
1100 1105 1110
Phe Ile Leu Ile Ile Gly Trp Val Phe Phe Met Thr Gly Cys Cys
1115 1120 1125
Gly Cys Cys Cys Gly Cys Phe Gly Ile Ile Pro Leu Met Asn Lys
1130 1135 1140
Cys Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Phe Asp Asn Asp Val
1145 1150 1155
Val Thr Glu Gln Tyr Arg Pro Lys Lys Ser Val
1160 1165
<210> 16
<211> 1082
<212> PRT
<213> avian infectious bronchitis Virus
<400> 16
Ser Leu Tyr Asn Asn Asp Ser Tyr Val Tyr Tyr Tyr Gln Ser Ala Phe
1 5 10 15
Arg Pro Phe Asn Gly Trp His Leu His Gly Gly Ala Tyr Ala Val Val
20 25 30
Asn Val Ser Gln Glu Thr Ala Asn Ala Gly Ser Ser Pro Ser Cys Thr
35 40 45
Ala Gly Ala Ile Tyr Trp Ser Lys Asn Phe Thr Ala Ser Ser Val Ala
50 55 60
Met Thr Ala Pro Leu Gln Gly Met Gln Trp Ser Thr Ile Gln Phe Cys
65 70 75 80
Thr Ala His Cys Asn Phe Thr Asn Ile Val Val Phe Val Thr His Cys
85 90 95
Tyr Lys Ser Gly Ser Thr Val Cys Pro Leu Thr Gly Leu Ile Pro Gln
100 105 110
Asn His Ile Arg Ile Ser Ala Met Lys Gln Gly Asn Asn Gly Pro Ser
115 120 125
Gly Leu Phe Tyr Asn Leu Thr Val Pro Val Thr Lys Tyr Ser Lys Phe
130 135 140
Lys Ser Leu Gln Cys Val Asn Asn Gln Thr Ser Val Tyr Leu Asn Gly
145 150 155 160
Asp Leu Val Phe Thr Ser Asn Glu Thr Lys Asp Val Ser Gly Ala Gly
165 170 175
Val Tyr Phe Lys Ala Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu
180 185 190
Val Lys Ala Leu Ala Tyr Phe Val Asn Gly Thr Ala His Asp Val Ile
195 200 205
Leu Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr
210 215 220
Gly Lys Phe Ser Asp Gly Phe Tyr Pro Phe Thr Asn Asp Thr Leu Val
225 230 235 240
Lys Glu Lys Phe Ile Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Leu
245 250 255
Thr Leu Thr Asn Phe Thr Phe Tyr Asn Glu Ser Asn Ala Leu Pro Asn
260 265 270
Asn Gly Gly Val Asp Thr Ile Gln Leu Tyr Gln Thr His Thr Ala Gln
275 280 285
Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Gln Tyr
290 295 300
Val Glu Ser Asn Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Gly Phe
305 310 315 320
Arg Pro Glu Ser Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val
325 330 335
Ser Leu Ala Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe
340 345 350
His Gly Arg Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly Pro Thr
355 360 365
Leu Cys Lys Gly Val Tyr Ser Gly Glu Leu Thr Arg Ser Tyr Gln Cys
370 375 380
Gly Leu Leu Val Phe Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr
385 390 395 400
Ser Thr Lys Pro Ile Val Leu Thr Gln His Asn Tyr Asn Asn Ile Thr
405 410 415
Leu Asp Arg Cys Val Glu Tyr Asn Ile Tyr Gly Arg Val Gly Gln Gly
420 425 430
Phe Ile Thr Asn Val Thr Glu Ser Ala Ala Ala Phe Asn Tyr Leu Glu
435 440 445
Asp Gly Gly Leu Ala Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe
450 455 460
Val Val Gln Gly Glu Tyr Gly Phe Asn Tyr Tyr Lys Val Asn Pro Cys
465 470 475 480
Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly Asn Leu Val Gly
485 490 495
Ile Leu Thr Ser Ile Asn Gln Thr Gly Ser Gln Ser Ile Glu Asn Gln
500 505 510
Phe Tyr Val Lys Leu Thr Asn Gly Ser Arg Arg Ser Arg Arg Ser Val
515 520 525
Ser Glu Asn Val Thr Ser Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys
530 535 540
Ile Lys Pro Asp Gly Ser Leu Ser Thr Ile Val Pro Lys Glu Leu Glu
545 550 555 560
Gln Phe Val Ala Pro Leu Leu Asn Val Thr Glu His Val Leu Ile Pro
565 570 575
Asp Ser Phe Asn Leu Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Met
580 585 590
Asp Lys Val Gln Ile Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Phe
595 600 605
Glu Cys Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile
610 615 620
Leu Ser Val Val Asn Ser Val Gly Gln Lys Glu Asp Met Glu Leu Leu
625 630 635 640
Ser Phe Tyr Ser Ser Thr Lys Pro Ser Gly Ile Ser Gln Pro Leu Phe
645 650 655
Asn Asn Phe Ser Thr Gly Asp Phe Asn Ile Ser Leu Leu Leu Thr Ser
660 665 670
Pro Ser Ser Pro Ser Gly Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr
675 680 685
Ser Val Glu Ser Val Gly Leu Pro Thr Asp Glu Ala Tyr Lys Lys Cys
690 695 700
Thr Ser Gly Pro Leu Gly Phe Val Lys Asp Leu Val Cys Ala Arg Glu
705 710 715 720
Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln
725 730 735
Thr Met Tyr Thr Ser Ser Leu Val Ala Ser Met Ala Leu Gly Gly Ile
740 745 750
Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile
755 760 765
Asn His Leu Gly Ile Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys
770 775 780
Ile Ala Ala Ser Phe Asn Arg Ala Ile Gly His Met Gln Glu Gly Phe
785 790 795 800
Lys Ser Thr Ser Leu Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys
805 810 815
Gln Ser Ala Ile Leu Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe
820 825 830
Gly Ala Ile Ser Ser Val Leu Gln Asp Ile Tyr Gln Gln Leu Asp Val
835 840 845
Ile Gln Ala Asp Ala Gln Val Asp Arg Ile Ile Thr Gly Arg Leu Ser
850 855 860
Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr Ile Ala Val
865 870 875 880
Ser Gln Gln Arg Ala Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys
885 890 895
Ser Gln Ser Thr Arg Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu
900 905 910
Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile Val Phe Ile His Phe Thr
915 920 925
Tyr Thr Pro Glu Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys
930 935 940
Val Lys Pro Pro Asn Ala Ser His Tyr Ala Ile Val Pro Val Asn Gly
945 950 955 960
Arg Gly Ile Phe Ile Gln Val Asn Gly Thr Tyr Tyr Ile Thr Ser Arg
965 970 975
Asp Met Tyr Met Pro Arg Asn Ile Thr Ala Gly Asp Ile Val Thr Leu
980 985 990
Thr Ser Cys Gln Ala Asn Tyr Val Ser Val Asn Lys Thr Val Ile Ser
995 1000 1005
Thr Phe Val Glu Asp Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser
1010 1015 1020
Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro Asp Phe Asp Glu
1025 1030 1035
Phe Asn Tyr Thr Ile Pro Val Leu Asn Ile Ser Asn Glu Ile Asp
1040 1045 1050
Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp
1055 1060 1065
Leu Glu Thr Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro
1070 1075 1080
<210> 17
<211> 3495
<212> DNA
<213> avian infectious bronchitis Virus
<400> 17
atgttggaca aaccgctttt actagtgact ctttggtatg cactatgtag tgctttgctc 60
tatgataata atacttacgt ttactactac caaagtgcct ttaggcctgg tccaggttgg 120
cacctatatg ggggtgctta tgcagtagat agggttttta atgaaaccaa caatgcaggc 180
agtgcatctg attgcactgc tggtactttt tatgaaagcc ataatatttc tgcttcttct 240
gtagccatga cagtaccaca taatggtatg tcttggtcag cttcacaatt ttgtacagct 300
cattgtaact tctcagactt tacagtgttc gttacgcatt gttttaaaaa tcaactcggt 360
agttgtccct tgacaggtat gattcctcag aatcatattc gtatttctgc tatgagagat 420
ggagttttgt tttataactt aacagttagc gtatctaaat accctagatt taaatcgctt 480
caatgtgtta gcaattctac atctgtctat gtaaatggtg accttgtttt cacttctaat 540
gaaacttctt acgttacggg tgcaggcgtt tattttaaaa gtggtgggcc tgtaacttat 600
aaagttatga aagaagttaa agccctagcc tactttatta atggtaccgc acaagaggtt 660
attttatgtg ataactcacc tagaggtttg cttgcatgtc agtataacac tggtaatttt 720
tcagatggat tctacccttt tactaatcat tctttagtta aggataggtt tattgtatat 780
cgagaaagta gcactaacac tactttaaag ttaactaatt tcagttttac taatgtaagt 840
aatgcttctc ctaattcagg tggcgttgat actttccaat tatatcaaac aagtactgct 900
caggatggtt attataattt taatttatca tttctgagta gttttgtgta taaaccatct 960
gattttatgt atgggtcata ccacccacat tgtaagttta gaccagagaa tattaataat 1020
ggcttatggt ttaattcatt atctgtgtca cttacttacg gacccattca aggtggttgt 1080
aagcaatctg tttttagtaa tagagcaact tgttgctatg cttattctta tcaagggcct 1140
agtagatgta agggtgttta tagaggggag ctaacgcaat actttgaatg tggacttcta 1200
gtttacgtaa ctaagagtga tggctctcgt atacaaacta gaagtgaacc actggtgtta 1260
actcaatata attataacaa cattacttta aataagtgtg ttgagtataa tatatatggt 1320
agggttggtc aaggttttat tactaatgta actgaagcaa ctgctaatta tagttatcta 1380
gcagatggtg gtttagctat tttagatacc tcaggagcca tagacatatt tgttgttcaa 1440
ggtgcatatg gtcttaatta ttataaggtt aatccctgtg aagatgttaa ccaacagttt 1500
gtagtgtctg gtggcaactt agttggcatt cttacatctc ataatgaaac aggttctgaa 1560
tctattgaga accagtttta catcaaactc actaacggaa cacgtcgctc tagacgttct 1620
gttactggga atgttacaaa ttgcccttat gttagttatg gcaagttttg tataaaacca 1680
gatggttctt tatctataat agtaccacaa gaattagaac agtttgtggc gcctttattc 1740
aatgttactg agcatgtgct catacctgat agttttaatt taactgtcac agatgagtac 1800
atacaaactc gtatggataa ggttcaaatt atttgccttc agtatgtttg tggtaattct 1860
attgaatgca gaaagttgtt tcagcagtat ggacctgttt gtgataatat attgtctgtt 1920
gtaaatggtg taggtcaaag agaggatatg gaacttttaa gtttctattc ttctactaaa 1980
cctagtggtt acaatacacc aatttttaat aatgttagca ctggtgactt taatatttcg 2040
ctcctactaa caccacctaa tagtcctact gggcgctctt ttattgaaga tcttctcttt 2100
acaagtgtag aatctgttgg attaccaact gatgaagagt ataaaaagtg tacagcagga 2160
cctttaggtt ttgttaaaga ccttgtttgt gctagagagt ataatggttt gctcgttctg 2220
cctcctatta ttactgcgga aatgcaaacc atgtatacta gttctttagt agcctctatg 2280
gctttaggtg gcattactgc agctggtgct ataccttttg ctacacaact gcaggccaga 2340
attaaccatt tgggtattac taattctctt ttgttgaaaa accaagaaaa aattgctgct 2400
tcctttaata aggccatcgg tcatatgcag gaagggttta aaagtacttc tctagcatta 2460
caacagattc aagatgttgt taataaacag agttctattc ttacagagac tatgcaatca 2520
cttaataaaa attttggtgc tatttcctct gtaattcaag acatttacca gcaactagat 2580
gctattcagg cagatgctca ggttgatcgt cttattacag gtagactctc ttcactatct 2640
gttttagctt ctgctaaaca ggcagagtat catagagtgt cacaacagcg tgagttggcc 2700
actcagaaaa ttaatgagtg tgttaagtct cagtctaata ggtattcatt ttgtggtaat 2760
ggtagacatg ttctaaccat accacagaat gcacccaatg gcatagtgtt tatacacttt 2820
acatacactc cagagagttt tgttaatgtt acggcaatag tagggttttg cgtaaaccca 2880
gctaatgcta gtcattatgc aatagtgcct gttaatggca ggggtgtttt tatagaagtt 2940
aatggtagtt actatatcac tgctcgtgat atgtatatgc caagagatat tactgcagga 3000
gacatagtca ctttgacttc ttgtcaagca aactatgtta atgtaaataa aaccgtcatt 3060
aacacttttg tggaagatga cgattttgat ttttatgatg aattgtcaaa atggtggaat 3120
gatactaagc atgagctacc agattttgat gaattcaatt ataccgttcc agttttaaat 3180
attagtaatg aaattgacag aattcaacag gttattcagg gattaaatga ttccctaata 3240
gaccttgaaa cactctcaat tctcaaaact tatattaaat ggccttggta tgtgtggctt 3300
gccattgcat tccttaccat tatttttatt ctggtacttt gttggatatt tttcatgacc 3360
ggttgttgcg gttgttgttg tggatgcttt ggtatcatac cgttaatgag taagtgtggt 3420
aagaaatctt cttactacac gacttttgat aatgatgtgg taacttaaca atacagacct 3480
aaaaagtctg tttaa 3495
<210> 18
<211> 3225
<212> DNA
<213> avian infectious bronchitis Virus
<400> 18
gctttgtatg actcgagttc ttacgtgtac tactaccaaa gtgccttcag accacctgat 60
ggttggcatt tacatggggg tgcgtatgcg gttgttaata tttctagtga atctaataat 120
gcaggctctt catctgggtg tactgttggt attattcatg gtggtcgtgt tgttaatgct 180
tcttctatag ctatgacggc accgtcatca ggtatggctt ggtctagcag tcagttttgt 240
actgcatact gtaacttttc agatactaca gtgtttgtta cacattgtta taaacatggt 300
gggtgtccta taactggcat gcttcaacag cattctatac gtgtttctgc tatgaaaaat 360
ggccagcttt tctataattt aacagttagt gtagctaagt accctacttt taaatcattt 420
cagtgtgtta ataatttaac atccgtatat ttaaatggtg atcttgttta cacctctaat 480
gagaccacag atgttacatc tgcaggtgtt tattttaaag ctggtggacc tataacttat 540
aaagttatga gagaagttag agccctggct tattttgtta atggtactgc acaagatgtt 600
attttgtgtg atgggtcacc tagaggcttg ttagcatgcc agtataatac tggcaatttt 660
tcagatggct tttatccttt tactaatagt agtttagtta agcagaagtt tattgtctat 720
cgtgaaaata gtgttaatac tacttttacg ttacacaatt tcacttttca taatgagact 780
ggcgccaacc caaatcctag tggtgtccag aatattcaaa cttaccaaac acaaacagct 840
cagagtggtt attataattt taatttttcc tttctgagta gttttgttta taaggagtct 900
aattttatgt atggatctta tcacccaagt tgtaatttta gactagaaac tattaataat 960
ggtttgtggt ttaattcact ttcagttagt attgcttacg gtcctcttca aggtggttgc 1020
aagcaatctg tctttagtgg tagagcaacc tgttgttatg cttactcata tggaggtcct 1080
ttgctgtgta aaggtgttta ttcaggtgag ttagatcata attttgaatg tggactgtta 1140
gtttatgtta ctaagagcgg tggctctcgt atacaaacag ccactgaacc gccagttata 1200
actcaacaca attataataa tattacttta aatacttgtg ttgattataa tatatatggc 1260
agaactggcc agggttttat tactaatgta accgactcag ctgttagtta taattatcta 1320
gcagacgcag gtttggctat tttagataca tctggttcca tagacatctt tgtcgtacaa 1380
agtgaatatg gtcttaatta ttataaggtt aacccttgcg aagatgtcaa ccagcagttt 1440
gtagtttctg gtggtaaatt agtaggtatt cttacttcac gtaatgagac tggttcccag 1500
cttcttgaga atcagtttta catcaaaatc actaatggaa cacgtcgttt tagacgttct 1560
attactgaaa gtgttgaaaa ttgcccttat gttagttatg gtaagttttg tataaaacct 1620
gatggcagta ttgccacaat agtaccaaaa cagttagaac agtttgtggc acctttactt 1680
aatgttactg aaaatgtgct catacctaac agttttaatt taactgttac agatgagtac 1740
atacaaactc ggatggataa ggtccaaatt aattgcctgc agtatatttg tggcaattct 1800
ctggagtgca gaaatttgtt tcaacaatat ggtcctgttt gcgacaacat attgtctgta 1860
gtaaatagtg ttggtcaaaa agaagatatg gaacttttga atttctattc ttctactaag 1920
ccggctggtt ttaatacacc agttcttagt aatgttagca ctggtgagtt taatattact 1980
ctttttttaa caacgcctag tagtcctaga aggcgttctt ttattgaaga ccttctattt 2040
acaagtgttg aatctgttgg attaccaaca gatgacgcat acaaaaattg cactgcaggt 2100
cctttaggct ttctgaaaga ccttgcatgt gctcgtgaat ataatggttt gcttgtgttg 2160
cctcctatta taacagcaga aatgcaaact ttgtatacaa gctctctagt agcttctatg 2220
gcttttggtg gtattactgc agctggtgct ataccttttg ccacacaact gcaggctaga 2280
attaatcact tgggtattac ccagtcactt cttttgaaga atcaagaaaa aattgctgct 2340
tcctttaata aggccatcgg tcatatgcag gaaggtttta gaagtacatc tttagcatta 2400
caacaaattc aagatgttgt taataagcag agtgctattc ttactgagac tatggcatca 2460
cttaataaaa attttggtgc catttcttct gtgattcaag aaatctacca gcaacttgac 2520
gccatacaag caaatgctca agtggatcgt cttataactg gtagattgtc atcactttct 2580
gttttagcat ctgctaagca ggcggagtat attagagtgt cacaacagcg tgagttagct 2640
actcagaaga ttaatgagtg tgttaagtca cagtccatta ggtactcctt ttgtggtaat 2700
ggacgacatg ttttaaccat accgcaaaat gcacctaatg gtatagtgtt tatacacttt 2760
tcttacactc cagatagttt tgttaatgtt actgcaatag tgggtttttg tgtaaagcca 2820
gctaatgcta gtcagtatgc aatagtaccc gctaatggta ggggtatttt tatacaagtt 2880
aatggtagtt actacatcac tgcacgagat atgtatatgc caagagctat tactgcagga 2940
gatatagtta cgcttacttc ttgtcaagca aattatgtaa gtgtaaataa gaccgtcatt 3000
actacattcg tagacaatga tgattttgat tttaatgacg aattgtcaaa atggtggaat 3060
gatactaagc atgagctacc agactttgac aaattcaatt acacagtacc tatacttgac 3120
attgatagtg aaattgatcg tattcaaggc gttatacagg gtcttaatga ctctctaata 3180
gaccttgaaa aactttcaat actcaaaact tatattaagt ggcct 3225
<210> 19
<211> 11398
<212> DNA
<213> Artificial sequence
<220>
<223> plasmid sequence having IBV
<400> 19
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420
tgcgtcgaga tcggccgccc gggtaatacg actcactata gggacttaag atagatatta 480
atatatatct attgcactag ccttgcgcta gatttcaaac ttaacaaaac ggacttaaat 540
acctacagct ggtccccata ggtgttccat tgcagtgcac tttagtgccc tggatggcac 600
ctggccacct gtcaggtttt tgttgttaaa atatcattgt tgctggtatc actgcttgtt 660
ttgccgtgtc tcactttata catccgttgc ttgggctacc tagtatccag cgtcctacgg 720
gcgccgtggt cggttcgagt gcgaaggacc tctggttcat ctagcggtag gcgggtgtgt 780
ggaagtagcg cttcagacgt actggttctg ttgcgtgaaa cgcggggtca cctcccccca 840
catacctcta agggcttttg agcctagcgt tgggctacgt tctcgcacaa ggtcggctat 900
acgacgtttg tagggggtag tgccaaacaa cccctgaggt gacaggttct ggtggtgttt 960
cgaaaacaac aatgtgtgtg ccgcataata tgcgcgttat gcattttgga gcaggtagtg 1020
ataaaggagt ggcgcctggt agtactgttc ttaaacagtg gctccccgaa ggaacactcc 1080
ttgtcgataa tgatattgta gattatgttt ctgacgcaca cgtttctgtg ctttcagatt 1140
gcaataaata taagacagag cacaagtttg atcttgtgat atctgatatg tatacagaca 1200
atgattcaaa aagaaagcat gaaggcgtga tagccaataa tggcaatgat gacgtcttca 1260
tatacctttc aagttttcta cgcaataatt tggctctggg aggcagtttt gctgtaaaat 1320
taacagagac aagttggcat gagagtttat atgacattgc acaggattgt gcatggtgga 1380
caatgttttg tacagcagtg aatgcatctt cttcagaagc attcctgatt ggtgttaatt 1440
acttgggtgc aagtgcaaag gttaaagtta gtggaaaaac actgcacgca aattatatat 1500
tttggaggaa ttgtaattat ttacaaacct ctgcttatag tatatttgat gttgctaagt 1560
ttgatttgag attgaaagca acgccagttg ttaatcttaa gactgaacaa aagacagact 1620
tagtctttaa tttgataaag tgtggtaagt tactggtaag agatgttggt aacacctctt 1680
ttactagtga ctcttttgtg tgcactatgt agtgctgctt tgtatgactc gagttcttac 1740
gtgtactact accaaagtgc cttcagacca cctgatggtt ggcatttaca tgggggtgcg 1800
tatgcggttg ttaatatttc tagtgaatct aataatgcag gctcttcatc tgggtgtact 1860
gttggtatta ttcatggtgg tcgtgttgtt aatgcttctt ctatagctat gacggcaccg 1920
tcatcaggta tggcttggtc tagcagtcag ttttgtactg catactgtaa cttttcagat 1980
actacagtgt ttgttacaca ttgttataaa catggtgggt gtcctataac tggcatgctt 2040
caacagcatt ctatacgtgt ttctgctatg aaaaatggcc agcttttcta taatttaaca 2100
gttagtgtag ctaagtaccc tacttttaaa tcatttcagt gtgttaataa tttaacatcc 2160
gtatatttaa atggtgatct tgtttacacc tctaatgaga ccacagatgt tacatctgca 2220
ggtgtttatt ttaaagctgg tggacctata acttataaag ttatgagaga agttagagcc 2280
ctggcttatt ttgttaatgg tactgcacaa gatgttattt tgtgtgatgg gtcacctaga 2340
ggcttgttag catgccagta taatactggc aatttttcag atggctttta tccttttact 2400
aatagtagtt tagttaagca gaagtttatt gtctatcgtg aaaatagtgt taatactact 2460
tttacgttac acaatttcac ttttcataat gagactggcg ccaacccaaa tcctagtggt 2520
gtccagaata ttcaaactta ccaaacacaa acagctcaga gtggttatta taattttaat 2580
ttttcctttc tgagtagttt tgtttataag gagtctaatt ttatgtatgg atcttatcac 2640
ccaagttgta attttagact agaaactatt aataatggtt tgtggtttaa ttcactttca 2700
gttagtattg cttacggtcc tcttcaaggt ggttgcaagc aatctgtctt tagtggtaga 2760
gcaacctgtt gttatgctta ctcatatgga ggtcctttgc tgtgtaaagg tgtttattca 2820
ggtgagttag atcataattt tgaatgtgga ctgttagttt atgttactaa gagcggtggc 2880
tctcgtatac aaacagccac tgaaccgcca gttataactc aacacaatta taataatatt 2940
actttaaata cttgtgttga ttataatata tatggcagaa ctggccaggg ttttattact 3000
aatgtaaccg actcagctgt tagttataat tatctagcag acgcaggttt ggctatttta 3060
gatacatctg gttccataga catctttgtc gtacaaagtg aatatggtct taattattat 3120
aaggttaacc cttgcgaaga tgtcaaccag cagtttgtag tttctggtgg taaattagta 3180
ggtattctta cttcacgtaa tgagactggt tcccagcttc ttgagaatca gttttacatc 3240
aaaatcacta atggaacacg tcgttttaga cgttctatta ctgaaagtgt tgaaaattgc 3300
ccttatgtta gttatggtaa gttttgtata aaacctgatg gcagtattgc cacaatagta 3360
ccaaaacagt tagaacagtt tgtggcacct ttacttaatg ttactgaaaa tgtgctcata 3420
cctaacagtt ttaatttaac tgttacagat gagtacatac aaactcggat ggataaggtc 3480
caaattaatt gcctgcagta tatttgtggc aattctctgg agtgcagaaa tttgtttcaa 3540
caatatggtc ctgtttgcga caacatattg tctgtagtaa atagtgttgg tcaaaaagaa 3600
gatatggaac ttttgaattt ctattcttct actaagccgg ctggttttaa tacaccagtt 3660
cttagtaatg ttagcactgg tgagtttaat attactcttt ttttaacaac gcctagtagt 3720
cctagaaggc gttcttttat tgaagacctt ctatttacaa gtgttgaatc tgttggatta 3780
ccaacagatg acgcatacaa aaattgcact gcaggtcctt taggctttct gaaagacctt 3840
gcatgtgctc gtgaatataa tggtttgctt gtgttgcctc ctattataac agcagaaatg 3900
caaactttgt atacaagctc tctagtagct tctatggctt ttggtggtat tactgcagct 3960
ggtgctatac cttttgccac acaactgcag gctagaatta atcacttggg tattacccag 4020
tcacttcttt tgaagaatca agaaaaaatt gctgcttcct ttaataaggc catcggtcat 4080
atgcaggaag gttttagaag tacatcttta gcattacaac aaattcaaga tgttgttaat 4140
aagcagagtg ctattcttac tgagactatg gcatcactta ataaaaattt tggtgccatt 4200
tcttctgtga ttcaagaaat ctaccagcaa cttgacgcca tacaagcaaa tgctcaagtg 4260
gatcgtctta taactggtag attgtcatca ctttctgttt tagcatctgc taagcaggcg 4320
gagtatatta gagtgtcaca acagcgtgag ttagctactc agaagattaa tgagtgtgtt 4380
aagtcacagt ccattaggta ctccttttgt ggtaatggac gacatgtttt aaccataccg 4440
caaaatgcac ctaatggtat agtgtttata cacttttctt acactccaga tagttttgtt 4500
aatgttactg caatagtggg tttttgtgta aagccagcta atgctagtca gtatgcaata 4560
gtacccgcta atggtagggg tatttttata caagttaatg gtagttacta catcactgca 4620
cgagatatgt atatgccaag agctattact gcaggagata tagttacgct tacttcttgt 4680
caagcaaatt atgtaagtgt aaataagacc gtcattacta cattcgtaga caatgatgat 4740
tttgatttta atgacgaatt gtcaaaatgg tggaatgata ctaagcatga gctaccagac 4800
tttgacaaat tcaattacac agtacctata cttgacattg atagtgaaat tgatcgtatt 4860
caaggcgtta tacagggtct taatgactct ctaatagacc ttgaaaaact ttcaatactc 4920
aaaacttata ttaagtggcc ttggtatgtg tggctagcca tagcttttgc cactattatc 4980
ttcatcttaa tattaggatg ggttttcttc atgactgggt gttgtggttg ttgttgtgga 5040
tgctttggca ttatgcctct aatgagtaag tgtggtaaga aatcttctta ttacacgact 5100
tttgataacg atgtggtaac tgaacaatac agacctaaaa agtctgttta atgatccaaa 5160
gtcccactag tttcttaata gtattaattt tgctttggtg taaacttgta ctaagttgtt 5220
ttagagagtt tattattgcc cttcaacaac taacacaagt tttactccaa attatcgata 5280
gtaatttaca gtctagactg accctttggc acagtctaga ctaatgttaa acttagaagc 5340
aattattgaa accggtgatc aagtgattca aaaaatcagt ttcaatttac agcatatttc 5400
aagtgtatta aacacagaag tatttgaccc ctttgactat tgttattaca gaggaggtaa 5460
tttttgggaa atagagtcag ctgaagattg ttcaggtgat gatgaattta ttgaataagt 5520
cgctagagga gaatggaagt tttctaacgg cactttacat atttgtagga tttttagcat 5580
tttatcttct aggtagagca cttcaagcat ttgtacaggc tgctgatgct tgttgtttat 5640
tttggtacac gtggttagta attccaggag ttaagggtac agcctttgta tacaagtata 5700
catatggtag aaaacttaac aattcggaat tagaagcagt tgttgttaac gagtttccta 5760
agaacggttg gaataataaa aatccagcaa attttcaaga tgtccaacga aacaaattgt 5820
actcttgact ttgaacagtc agttgagctt tttaaagagt ataatttatt tataactgca 5880
ttcttgttgt tcttaaccat aatacttcag tatggttatg caacgcgtag taagtttatt 5940
tatatactta aaatgatagt gttatggtgc ttttggcccc ttaacattgc agtaggtgta 6000
atttcatgta tatacccacc aaacacagga ggtcttgtcg cagcgataat acttactgtg 6060
tttgcgtgtc tttcttttgt aggttattgg atccagagta ttagactctt taagcggtgt 6120
agatcttggt ggtcatttaa cccagaatct aacgccgtag gttcaatact cctaactaat 6180
ggtcaacaat gtaattttgc tatagagagt gtgccgatgg tgctttctcc tattataaag 6240
aatggtgttc tttattgtga gggtcagtgg cttgctaaat gtgaaccaga ccacttgcct 6300
aaagacatat ttgtatgcac accagataga cgtaatatct atcgtatggt gcagaaatac 6360
actggtgacc aaagcggaaa taagaaaagg tttgctacat ttgtctatgc aaagcagtca 6420
gtagacactg gcgagctaga aagtgtagca acaggtggaa gtagccttta cacataaatg 6480
tgtgtgtgta gagagtattt aaaattattc ttcaatagtg cctctatttt aagagcgcgg 6540
aagagtattt gttttgagga tattaatata aatcctcttt gttttgtact ctctttacaa 6600
gagttattat ttaagcaaca gtttttcctt tcctttgttt ggaagaaagt tgttgttaat 6660
ggtgtagaat tccaagtaga aaatggaaaa gtccactacg aaggaaaccc cattttccaa 6720
aaaggttgtt gtaggttgtg gtcccattat aagaaggatt aaatggatta aaccacctac 6780
actacttact tgtaataagg gcgtttggac ttacaagcgc ttaacaaata cagacgatga 6840
aatggctgac tagttttgga agagcagtta tttcttgtta taaagcccta ctattaactc 6900
agttaagagt attagatagg ttaattttag atcacggacc aaagcgagtc ttaacgtgtg 6960
gtaggcgagt gcttttatct caattagatt tagtttatag gttggcatat acgcccaccc 7020
aatcgctggt atgaataata gtaaagataa tccttttcgc ggagcaatag caagaaaagc 7080
gcgaatttat ctgagagaag gattagagtg tgtttacttt cttaacaaag caggacaagc 7140
agagccttgt cccgcgtgta cctccctagt atttcagggg aaaacttgtg aggaacacac 7200
agataataat aatcttttgt catggcgagc ggtaagacaa ctgggaagac agacgcccca 7260
gcgccagtca tcaaactagg agggccaaaa ccacctaaag ttggttcttc tggaaatgct 7320
agctggtttc aagcactaaa agccaagaag ttaaattcac ctcctcctaa gtttgaaggt 7380
agcggcgttc ctgataatga aaatcttaaa ttaagccagc aacatgggta ctggagacgt 7440
caagccaggt acaagccagg taaaggcgga agaaaatcag tcccagatgc ttggtacttc 7500
tattacactg gaacaggacc agccgctgac ctgaattggg gtgatagcca agatggtata 7560
gtgtgggttt ctgcaaaggg tgctgatact aaatctagat ctaaccaggg tacaagggat 7620
cctgataagt ttgaccaata cccgctacga ttctcagatg gaggacctga tggtaatttc 7680
cgttgggact tcattccaat aaatcgtggt aggagtggaa gatcaacagc ggcttcatca 7740
gcagcatcta gtagagcacc gtcgcgtgat ggctcgcgtg gacgtagaag cggagctgaa 7800
gatgatctta tagctcgtgc agcaaagatc attcaggatc agcagaagaa gggttctcgc 7860
attactaaag ctaaggccga tgaaatggct catcgccggt attgtaagcg tactatccca 7920
cctggttata aggttgatca agtatttggt ccccgtacta aaggtaagga gggaaatttt 7980
ggtgatgaca agatgaatga ggagggtatt aaggatgggc gcgttacagc aatgctcaac 8040
ctagtcccta gcagccatgc ttgtcttttt ggaagtagag tgacgcccaa acttcaacca 8100
gatgggctgc acttgagatt tgaatttact actgtggttt ctagggatga tccgcagttt 8160
gataattatg tgaaaatttg tgatcagtgt gtcgatggtg tagggactcg gccaaaagac 8220
gatgaaccga gaccaaagtc acgcccaaat tcaagacctg ctacaagaac aagttctcca 8280
gcgccaagac aacagcgtca aaagaaggag aagaagtcaa agaagcagga tgatgaagta 8340
gataaggcat tgacctcaga tgaggagagg aacaatgcac agctggaatt tgatgatgaa 8400
ccgaaagtga ttaactgggg ggattcagca cttggagaga atgagttgta aagctagatt 8460
tccaacttaa catcatggac gtgcgtatgc tgtttttccc tactatagac tttttagcat 8520
attatttttt gctatttgta tggtttatta caggtgaaga ttgtatgtat ttgttgtaca 8580
ctcgtatgtt ctatattatg ttttctgtag ttgttattag tgttgttctt gttcttactc 8640
tactgttctc ttttctttat tttagagtat caataagaat caaggaagat aggcatgtag 8700
tttgattacc tacatgtcta tcgccaggga aatgtctaat ctgtctactt agtagcctgg 8760
aaacgaacgg tagaccctta gattttaatt tagtttaatt tttagtttag tttaagttag 8820
tttagagtag gtataaagaa gccagtgccg gggccacgcg gagtacgatc gagggtacag 8880
cactaggacg cccactaggg gaagagctaa attttagttt aagttaagtt taattggcta 8940
agtatagtta aaatttatag gctagtatag agttagagca aaaaaaaaaa aaaaaaaaaa 9000
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9060
aaaaaaaaaa aaaaaaaaag tttaaactta attaagaatt cccttggctc gagttcgaaa 9120
tcggatgccg ggaccgacga gtgcagaggc gtgcaagcga gcttggcgta atcatggtca 9180
tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 9240
agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 9300
cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 9360
caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 9420
tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 9480
cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 9540
aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 9600
gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 9660
agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 9720
cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 9780
cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 9840
ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 9900
gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 9960
tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 10020
acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 10080
tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 10140
attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 10200
gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 10260
ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 10320
taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 10380
ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 10440
ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 10500
gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 10560
ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 10620
gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 10680
tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 10740
atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 10800
gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 10860
tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 10920
atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 10980
agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 11040
ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 11100
tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 11160
aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 11220
tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 11280
aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 11340
accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 11398
<210> 20
<211> 3231
<212> DNA
<213> avian infectious bronchitis Virus
<400> 20
ttgctctatg ataataatac ttacgtttac tactaccaaa gtgcctttag gcctggtcca 60
ggttggcacc tatatggggg tgcttatgca gtagataggg tttttaatga aaccaacaat 120
gcaggcagtg catctgattg cactgctggt actttttatg aaagccataa tatttctgct 180
tcttctgtag ccatgacagt accacataat ggtatgtctt ggtcagcttc acaattttgt 240
acagctcatt gtaacttctc agactttaca gtgttcgtta cgcattgttt taaaaatcaa 300
ctcggtagtt gtcccttgac aggtatgatt cctcagaatc atattcgtat ttctgctatg 360
agagatggag ttttgtttta taacttaaca gttagcgtat ctaaataccc tagatttaaa 420
tcgcttcaat gtgttagcaa ttctacatct gtctatgtaa atggtgacct tgttttcact 480
tctaatgaaa cttcttacgt tacgggtgca ggcgtttatt ttaaaagtgg tgggcctgta 540
acttataaag ttatgaaaga agttaaagcc ctagcctact ttattaatgg taccgcacaa 600
gaggttattt tatgtgataa ctcacctaga ggtttgcttg catgtcagta taacactggt 660
aatttttcag atggattcta cccttttact aatcattctt tagttaagga taggtttatt 720
gtatatcgag aaagtagcac taacactact ttaaagttaa ctaatttcag ttttactaat 780
gtaagtaatg cttctcctaa ttcaggtggc gttgatactt tccaattata tcaaacaagt 840
actgctcagg atggttatta taattttaat ttatcatttc tgagtagttt tgtgtataaa 900
ccatctgatt ttatgtatgg gtcataccac ccacattgta agtttagacc agagaatatt 960
aataatggct tatggtttaa ttcattatct gtgtcactta cttacggacc cattcaaggt 1020
ggttgtaagc aatctgtttt tagtaataga gcaacttgtt gctatgctta ttcttatcaa 1080
gggcctagta gatgtaaggg tgtttataga ggggagctaa cgcaatactt tgaatgtgga 1140
cttctagttt acgtaactaa gagtgatggc tctcgtatac aaactagaag tgaaccactg 1200
gtgttaactc aatataatta taacaacatt actttaaata agtgtgttga gtataatata 1260
tatggtaggg ttggtcaagg ttttattact aatgtaactg aagcaactgc taattatagt 1320
tatctagcag atggtggttt agctatttta gatacctcag gagccataga catatttgtt 1380
gttcaaggtg catatggtct taattattat aaggttaatc cctgtgaaga tgttaaccaa 1440
cagtttgtag tgtctggtgg caacttagtt ggcattctta catctcataa tgaaacaggt 1500
tctgaatcta ttgagaacca gttttacatc aaactcacta acggaacacg tcgctctaga 1560
cgttctgtta ctgggaatgt tacaaattgc ccttatgtta gttatggcaa gttttgtata 1620
aaaccagatg gttctttatc tataatagta ccacaagaat tagaacagtt tgtggcgcct 1680
ttattcaatg ttactgagca tgtgctcata cctgatagtt ttaatttaac tgtcacagat 1740
gagtacatac aaactcgtat ggataaggtt caaattattt gccttcagta tgtttgtggt 1800
aattctattg aatgcagaaa gttgtttcag cagtatggac ctgtttgtga taatatattg 1860
tctgttgtaa atggtgtagg tcaaagagag gatatggaac ttttaagttt ctattcttct 1920
actaaaccta gtggttacaa tacaccaatt tttaataatg ttagcactgg tgactttaat 1980
atttcgctcc tactaacacc acctaatagt cctactgggc gctcttttat tgaagatctt 2040
ctctttacaa gtgtagaatc tgttggatta ccaactgatg aagagtataa aaagtgtaca 2100
gcaggacctt taggttttgt taaagacctt gtttgtgcta gagagtataa tggtttgctc 2160
gttctgcctc ctattattac tgcggaaatg caaaccatgt atactagttc tttagtagcc 2220
tctatggctt taggtggcat tactgcagct ggtgctatac cttttgctac acaactgcag 2280
gccagaatta accatttggg tattactaat tctcttttgt tgaaaaacca agaaaaaatt 2340
gctgcttcct ttaataaggc catcggtcat atgcaggaag ggtttaaaag tacttctcta 2400
gcattacaac agattcaaga tgttgttaat aaacagagtt ctattcttac agagactatg 2460
caatcactta ataaaaattt tggtgctatt tcctctgtaa ttcaagacat ttaccagcaa 2520
ctagatgcta ttcaggcaga tgctcaggtt gatcgtctta ttacaggtag actctcttca 2580
ctatctgttt tagcttctgc taaacaggca gagtatcata gagtgtcaca acagcgtgag 2640
ttggccactc agaaaattaa tgagtgtgtt aagtctcagt ctaataggta ttcattttgt 2700
ggtaatggta gacatgttct aaccatacca cagaatgcac ccaatggcat agtgtttata 2760
cactttacat acactccaga gagttttgtt aatgttacgg caatagtagg gttttgcgta 2820
aacccagcta atgctagtca ttatgcaata gtgcctgtta atggcagggg tgtttttata 2880
gaagttaatg gtagttacta tatcactgct cgtgatatgt atatgccaag agatattact 2940
gcaggagaca tagtcacttt gacttcttgt caagcaaact atgttaatgt aaataaaacc 3000
gtcattaaca cttttgtgga agatgacgat tttgattttt atgatgaatt gtcaaaatgg 3060
tggaatgata ctaagcatga gctaccagat tttgatgaat tcaattatac cgttccagtt 3120
ttaaatatta gtaatgaaat tgacagaatt caacaggtta ttcagggatt aaatgattcc 3180
ctaatagacc ttgaaacact ctcaattctc aaaacttata ttaaatggcc t 3231
<210> 21
<211> 11404
<212> DNA
<213> Artificial sequence
<220>
<223> plasmid having IBV
<400> 21
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420
tgcgtcgaga tcggccgccc gggtaatacg actcactata gggacttaag atagatatta 480
atatatatct attgcactag ccttgcgcta gatttcaaac ttaacaaaac ggacttaaat 540
acctacagct ggtccccata ggtgttccat tgcagtgcac tttagtgccc tggatggcac 600
ctggccacct gtcaggtttt tgttgttaaa atatcattgt tgctggtatc actgcttgtt 660
ttgccgtgtc tcactttata catccgttgc ttgggctacc tagtatccag cgtcctacgg 720
gcgccgtggt cggttcgagt gcgaaggacc tctggttcat ctagcggtag gcgggtgtgt 780
ggaagtagcg cttcagacgt actggttctg ttgcgtgaaa cgcggggtca cctcccccca 840
catacctcta agggcttttg agcctagcgt tgggctacgt tctcgcacaa ggtcggctat 900
acgacgtttg tagggggtag tgccaaacaa cccctgaggt gacaggttct ggtggtgttt 960
cgaaaacaac aatgtgtgtg ccgcataata tgcgcgttat gcattttgga gcaggtagtg 1020
ataaaggagt ggcgcctggt agtactgttc ttaaacagtg gctccccgaa ggaacactcc 1080
ttgtcgataa tgatattgta gattatgttt ctgacgcaca cgtttctgtg ctttcagatt 1140
gcaataaata taagacagag cacaagtttg atcttgtgat atctgatatg tatacagaca 1200
atgattcaaa aagaaagcat gaaggcgtga tagccaataa tggcaatgat gacgtcttca 1260
tatacctttc aagttttcta cgcaataatt tggctctggg aggcagtttt gctgtaaaat 1320
taacagagac aagttggcat gagagtttat atgacattgc acaggattgt gcatggtgga 1380
caatgttttg tacagcagtg aatgcatctt cttcagaagc attcctgatt ggtgttaatt 1440
acttgggtgc aagtgcaaag gttaaagtta gtggaaaaac actgcacgca aattatatat 1500
tttggaggaa ttgtaattat ttacaaacct ctgcttatag tatatttgat gttgctaagt 1560
ttgatttgag attgaaagca acgccagttg ttaatcttaa gactgaacaa aagacagact 1620
tagtctttaa tttgataaag tgtggtaagt tactggtaag agatgttggt aacacctctt 1680
ttactagtga ctcttttgtg tgcactatgt agtgctttgc tctatgataa taatacttac 1740
gtttactact accaaagtgc ctttaggcct ggtccaggtt ggcacctata tgggggtgct 1800
tatgcagtag atagggtttt taatgaaacc aacaatgcag gcagtgcatc tgattgcact 1860
gctggtactt tttatgaaag ccataatatt tctgcttctt ctgtagccat gacagtacca 1920
cataatggta tgtcttggtc agcttcacaa ttttgtacag ctcattgtaa cttctcagac 1980
tttacagtgt tcgttacgca ttgttttaaa aatcaactcg gtagttgtcc cttgacaggt 2040
atgattcctc agaatcatat tcgtatttct gctatgagag atggagtttt gttttataac 2100
ttaacagtta gcgtatctaa ataccctaga tttaaatcgc ttcaatgtgt tagcaattct 2160
acatctgtct atgtaaatgg tgaccttgtt ttcacttcta atgaaacttc ttacgttacg 2220
ggtgcaggcg tttattttaa aagtggtggg cctgtaactt ataaagttat gaaagaagtt 2280
aaagccctag cctactttat taatggtacc gcacaagagg ttattttatg tgataactca 2340
cctagaggtt tgcttgcatg tcagtataac actggtaatt tttcagatgg attctaccct 2400
tttactaatc attctttagt taaggatagg tttattgtat atcgagaaag tagcactaac 2460
actactttaa agttaactaa tttcagtttt actaatgtaa gtaatgcttc tcctaattca 2520
ggtggcgttg atactttcca attatatcaa acaagtactg ctcaggatgg ttattataat 2580
tttaatttat catttctgag tagttttgtg tataaaccat ctgattttat gtatgggtca 2640
taccacccac attgtaagtt tagaccagag aatattaata atggcttatg gtttaattca 2700
ttatctgtgt cacttactta cggacccatt caaggtggtt gtaagcaatc tgtttttagt 2760
aatagagcaa cttgttgcta tgcttattct tatcaagggc ctagtagatg taagggtgtt 2820
tatagagggg agctaacgca atactttgaa tgtggacttc tagtttacgt aactaagagt 2880
gatggctctc gtatacaaac tagaagtgaa ccactggtgt taactcaata taattataac 2940
aacattactt taaataagtg tgttgagtat aatatatatg gtagggttgg tcaaggtttt 3000
attactaatg taactgaagc aactgctaat tatagttatc tagcagatgg tggtttagct 3060
attttagata cctcaggagc catagacata tttgttgttc aaggtgcata tggtcttaat 3120
tattataagg ttaatccctg tgaagatgtt aaccaacagt ttgtagtgtc tggtggcaac 3180
ttagttggca ttcttacatc tcataatgaa acaggttctg aatctattga gaaccagttt 3240
tacatcaaac tcactaacgg aacacgtcgc tctagacgtt ctgttactgg gaatgttaca 3300
aattgccctt atgttagtta tggcaagttt tgtataaaac cagatggttc tttatctata 3360
atagtaccac aagaattaga acagtttgtg gcgcctttat tcaatgttac tgagcatgtg 3420
ctcatacctg atagttttaa tttaactgtc acagatgagt acatacaaac tcgtatggat 3480
aaggttcaaa ttatttgcct tcagtatgtt tgtggtaatt ctattgaatg cagaaagttg 3540
tttcagcagt atggacctgt ttgtgataat atattgtctg ttgtaaatgg tgtaggtcaa 3600
agagaggata tggaactttt aagtttctat tcttctacta aacctagtgg ttacaataca 3660
ccaattttta ataatgttag cactggtgac tttaatattt cgctcctact aacaccacct 3720
aatagtccta ctgggcgctc ttttattgaa gatcttctct ttacaagtgt agaatctgtt 3780
ggattaccaa ctgatgaaga gtataaaaag tgtacagcag gacctttagg ttttgttaaa 3840
gaccttgttt gtgctagaga gtataatggt ttgctcgttc tgcctcctat tattactgcg 3900
gaaatgcaaa ccatgtatac tagttcttta gtagcctcta tggctttagg tggcattact 3960
gcagctggtg ctataccttt tgctacacaa ctgcaggcca gaattaacca tttgggtatt 4020
actaattctc ttttgttgaa aaaccaagaa aaaattgctg cttcctttaa taaggccatc 4080
ggtcatatgc aggaagggtt taaaagtact tctctagcat tacaacagat tcaagatgtt 4140
gttaataaac agagttctat tcttacagag actatgcaat cacttaataa aaattttggt 4200
gctatttcct ctgtaattca agacatttac cagcaactag atgctattca ggcagatgct 4260
caggttgatc gtcttattac aggtagactc tcttcactat ctgttttagc ttctgctaaa 4320
caggcagagt atcatagagt gtcacaacag cgtgagttgg ccactcagaa aattaatgag 4380
tgtgttaagt ctcagtctaa taggtattca ttttgtggta atggtagaca tgttctaacc 4440
ataccacaga atgcacccaa tggcatagtg tttatacact ttacatacac tccagagagt 4500
tttgttaatg ttacggcaat agtagggttt tgcgtaaacc cagctaatgc tagtcattat 4560
gcaatagtgc ctgttaatgg caggggtgtt tttatagaag ttaatggtag ttactatatc 4620
actgctcgtg atatgtatat gccaagagat attactgcag gagacatagt cactttgact 4680
tcttgtcaag caaactatgt taatgtaaat aaaaccgtca ttaacacttt tgtggaagat 4740
gacgattttg atttttatga tgaattgtca aaatggtgga atgatactaa gcatgagcta 4800
ccagattttg atgaattcaa ttataccgtt ccagttttaa atattagtaa tgaaattgac 4860
agaattcaac aggttattca gggattaaat gattccctaa tagaccttga aacactctca 4920
attctcaaaa cttatattaa atggccttgg tatgtgtggc tagccatagc ttttgccact 4980
attatcttca tcttaatatt aggatgggtt ttcttcatga ctgggtgttg tggttgttgt 5040
tgtggatgct ttggcattat gcctctaatg agtaagtgtg gtaagaaatc ttcttattac 5100
acgacttttg ataacgatgt ggtaactgaa caatacagac ctaaaaagtc tgtttaatga 5160
tccaaagtcc cactagtttc ttaatagtat taattttgct ttggtgtaaa cttgtactaa 5220
gttgttttag agagtttatt attgcccttc aacaactaac acaagtttta ctccaaatta 5280
tcgatagtaa tttacagtct agactgaccc tttggcacag tctagactaa tgttaaactt 5340
agaagcaatt attgaaaccg gtgatcaagt gattcaaaaa atcagtttca atttacagca 5400
tatttcaagt gtattaaaca cagaagtatt tgaccccttt gactattgtt attacagagg 5460
aggtaatttt tgggaaatag agtcagctga agattgttca ggtgatgatg aatttattga 5520
ataagtcgct agaggagaat ggaagttttc taacggcact ttacatattt gtaggatttt 5580
tagcatttta tcttctaggt agagcacttc aagcatttgt acaggctgct gatgcttgtt 5640
gtttattttg gtacacgtgg ttagtaattc caggagttaa gggtacagcc tttgtataca 5700
agtatacata tggtagaaaa cttaacaatt cggaattaga agcagttgtt gttaacgagt 5760
ttcctaagaa cggttggaat aataaaaatc cagcaaattt tcaagatgtc caacgaaaca 5820
aattgtactc ttgactttga acagtcagtt gagcttttta aagagtataa tttatttata 5880
actgcattct tgttgttctt aaccataata cttcagtatg gttatgcaac gcgtagtaag 5940
tttatttata tacttaaaat gatagtgtta tggtgctttt ggccccttaa cattgcagta 6000
ggtgtaattt catgtatata cccaccaaac acaggaggtc ttgtcgcagc gataatactt 6060
actgtgtttg cgtgtctttc ttttgtaggt tattggatcc agagtattag actctttaag 6120
cggtgtagat cttggtggtc atttaaccca gaatctaacg ccgtaggttc aatactccta 6180
actaatggtc aacaatgtaa ttttgctata gagagtgtgc cgatggtgct ttctcctatt 6240
ataaagaatg gtgttcttta ttgtgagggt cagtggcttg ctaaatgtga accagaccac 6300
ttgcctaaag acatatttgt atgcacacca gatagacgta atatctatcg tatggtgcag 6360
aaatacactg gtgaccaaag cggaaataag aaaaggtttg ctacatttgt ctatgcaaag 6420
cagtcagtag acactggcga gctagaaagt gtagcaacag gtggaagtag cctttacaca 6480
taaatgtgtg tgtgtagaga gtatttaaaa ttattcttca atagtgcctc tattttaaga 6540
gcgcggaaga gtatttgttt tgaggatatt aatataaatc ctctttgttt tgtactctct 6600
ttacaagagt tattatttaa gcaacagttt ttcctttcct ttgtttggaa gaaagttgtt 6660
gttaatggtg tagaattcca agtagaaaat ggaaaagtcc actacgaagg aaaccccatt 6720
ttccaaaaag gttgttgtag gttgtggtcc cattataaga aggattaaat ggattaaacc 6780
acctacacta cttacttgta ataagggcgt ttggacttac aagcgcttaa caaatacaga 6840
cgatgaaatg gctgactagt tttggaagag cagttatttc ttgttataaa gccctactat 6900
taactcagtt aagagtatta gataggttaa ttttagatca cggaccaaag cgagtcttaa 6960
cgtgtggtag gcgagtgctt ttatctcaat tagatttagt ttataggttg gcatatacgc 7020
ccacccaatc gctggtatga ataatagtaa agataatcct tttcgcggag caatagcaag 7080
aaaagcgcga atttatctga gagaaggatt agagtgtgtt tactttctta acaaagcagg 7140
acaagcagag ccttgtcccg cgtgtacctc cctagtattt caggggaaaa cttgtgagga 7200
acacacagat aataataatc ttttgtcatg gcgagcggta agacaactgg gaagacagac 7260
gccccagcgc cagtcatcaa actaggaggg ccaaaaccac ctaaagttgg ttcttctgga 7320
aatgctagct ggtttcaagc actaaaagcc aagaagttaa attcacctcc tcctaagttt 7380
gaaggtagcg gcgttcctga taatgaaaat cttaaattaa gccagcaaca tgggtactgg 7440
agacgtcaag ccaggtacaa gccaggtaaa ggcggaagaa aatcagtccc agatgcttgg 7500
tacttctatt acactggaac aggaccagcc gctgacctga attggggtga tagccaagat 7560
ggtatagtgt gggtttctgc aaagggtgct gatactaaat ctagatctaa ccagggtaca 7620
agggatcctg ataagtttga ccaatacccg ctacgattct cagatggagg acctgatggt 7680
aatttccgtt gggacttcat tccaataaat cgtggtagga gtggaagatc aacagcggct 7740
tcatcagcag catctagtag agcaccgtcg cgtgatggct cgcgtggacg tagaagcgga 7800
gctgaagatg atcttatagc tcgtgcagca aagatcattc aggatcagca gaagaagggt 7860
tctcgcatta ctaaagctaa ggccgatgaa atggctcatc gccggtattg taagcgtact 7920
atcccacctg gttataaggt tgatcaagta tttggtcccc gtactaaagg taaggaggga 7980
aattttggtg atgacaagat gaatgaggag ggtattaagg atgggcgcgt tacagcaatg 8040
ctcaacctag tccctagcag ccatgcttgt ctttttggaa gtagagtgac gcccaaactt 8100
caaccagatg ggctgcactt gagatttgaa tttactactg tggtttctag ggatgatccg 8160
cagtttgata attatgtgaa aatttgtgat cagtgtgtcg atggtgtagg gactcggcca 8220
aaagacgatg aaccgagacc aaagtcacgc ccaaattcaa gacctgctac aagaacaagt 8280
tctccagcgc caagacaaca gcgtcaaaag aaggagaaga agtcaaagaa gcaggatgat 8340
gaagtagata aggcattgac ctcagatgag gagaggaaca atgcacagct ggaatttgat 8400
gatgaaccga aagtgattaa ctggggggat tcagcacttg gagagaatga gttgtaaagc 8460
tagatttcca acttaacatc atggacgtgc gtatgctgtt tttccctact atagactttt 8520
tagcatatta ttttttgcta tttgtatggt ttattacagg tgaagattgt atgtatttgt 8580
tgtacactcg tatgttctat attatgtttt ctgtagttgt tattagtgtt gttcttgttc 8640
ttactctact gttctctttt ctttatttta gagtatcaat aagaatcaag gaagataggc 8700
atgtagtttg attacctaca tgtctatcgc cagggaaatg tctaatctgt ctacttagta 8760
gcctggaaac gaacggtaga cccttagatt ttaatttagt ttaattttta gtttagttta 8820
agttagttta gagtaggtat aaagaagcca gtgccggggc cacgcggagt acgatcgagg 8880
gtacagcact aggacgccca ctaggggaag agctaaattt tagtttaagt taagtttaat 8940
tggctaagta tagttaaaat ttataggcta gtatagagtt agagcaaaaa aaaaaaaaaa 9000
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9060
aaaaaaaaaa aaaaaaaaaa aaaaagttta aacttaatta agaattccct tggctcgagt 9120
tcgaaatcgg atgccgggac cgacgagtgc agaggcgtgc aagcgagctt ggcgtaatca 9180
tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 9240
gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 9300
gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 9360
atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 9420
actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 9480
gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 9540
cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 9600
ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 9660
ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 9720
ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 9780
agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 9840
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 9900
aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 9960
gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 10020
agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 10080
ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 10140
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 10200
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 10260
aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 10320
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 10380
atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 10440
cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 10500
gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 10560
gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 10620
tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 10680
tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 10740
tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 10800
aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 10860
atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 10920
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 10980
catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 11040
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 11100
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 11160
gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 11220
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 11280
tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 11340
taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 11400
cgtc 11404
<210> 22
<211> 52
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 22
cagagcacaa gtttgatctt gtgatatctg atatgtatac agacaatgat tc 52
<210> 23
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 23
tatcatagag caaagcacta catagtgcac ac 32
<210> 24
<211> 38
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 24
actatgtagt gctttgctct atgataataa tacttacg 38
<210> 25
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 25
cataccaagg ccatttaata taagttttga gaattgagag 40
<210> 26
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 26
aacttatatt aaatggcctt ggtatgtgtg g 31
<210> 27
<211> 48
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 27
cttaactcct ggaattacta accacgtgta ccaaaataaa caacaagc 48
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 28
tcagcatgga cgtgtggtta 20
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 29
aggttggcac ctatatgggg 20
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 30
tgacttggtt tgaagatggc 20
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 31
aagagatgtt ggtaacacct 20
<210> 32
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 32
gacagagcac aagtttgatc 20
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 33
ggagtgaaaa caagatcacc 20
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 34
aatttaacag ttagcgtatc 20
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 35
tttgtatacg agagccatca 20
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 36
ggtcctacta gatgtaaggg 20
<210> 37
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 37
ctctctttga cctacaccat 20
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 38
ttgccttcag tatgtttgtg 20
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 39
agtgaagaaa gtctacctgt 20
<210> 40
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 40
atttcctccg tacttcaaga 20
<210> 41
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 41
tgaagataat aatggcaaaa gc 22
<210> 42
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 42
tcttgaaaca ctctcaattc t 21
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 43
ggtcaccagt atatttctgc 20
<210> 44
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 44
ggtcaacaat gtaattttgc t 21
<210> 45
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 45
cttgtcctgc tttgttaaga 20
<210> 46
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 46
ttataggttg gcttgtacgc 20
<210> 47
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 47
gcccatcctt aataccttcc 20
<210> 48
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 48
ctcgcattac aaaggctaag 20
<210> 49
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 49
gctctaactc tatactagcc t 21
<210> 50
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 50
gtttgtccaa catctcttac cagtaactta cc 32
<210> 51
<211> 35
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 51
ttactggtaa gagatgttgg acaaaccgct tttac 35
<210> 52
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 52
ggactttgga tcattaaaca gactttttag gtctgtattg 40
<210> 53
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 53
aaagtctgtt taatgatcca aagtcccact ag 32
<210> 54
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 54
acttcaccaa catctcttac cagtaactta cc 32
<210> 55
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 55
ttactggtaa gagatgttgg tgaagtcact g 31
<210> 56
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 56
aatcaaacaa attagcacta catagtgcac ac 32
<210> 57
<211> 41
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 57
actatgtagt gctaatttgt ttgattctga taataattat g 41
<210> 58
<211> 46
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 58
cataccaagg ccacttaata taagttttaa ttattgaaag ttcttc 46
<210> 59
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 59
aacttatatt aagtggcctt ggtatgtgtg g 31
<210> 60
<211> 48
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 60
cagagcacaa gtttgatctt gtgatatctg atatgtatac agacaatg 48
<210> 61
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 61
acttccccaa catctcttac cagtaactta cc 32
<210> 62
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 62
ttactggtaa gagatgttgg ggaagtcact g 31
<210> 63
<211> 47
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 63
cttaactcct ggaattacta accacgtgta ccaaaataaa caacaag 47
<210> 64
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 64
tatcaaacaa agcagcacta catagtgcac ac 32
<210> 65
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 65
actatgtagt gctgctttgt ttgataataa tgaaac 36
<210> 66
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 66
cataccaagg ccatttaata taagtcttga gtattgaaag 40
<210> 67
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 67
gacttatatt aaatggcctt ggtatgtgtg g 31
<210> 68
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 68
tgtcatataa attagcacta catagtgcac ac 32
<210> 69
<211> 38
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 69
actatgtagt gctaatttat atgacaacga atcttttg 38
<210> 70
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 70
cataccaagg ccacttaata taagttttga gtattgaaag 40
<210> 71
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 71
tatcaaacag agcagcacta catagtgcac ac 32
<210> 72
<211> 34
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 72
actatgtagt gctgctctgt ttgataataa tcag 34
<210> 73
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 73
gttgaaccaa catctcttac cagtaactta cc 32
<210> 74
<211> 35
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 74
ttactggtaa gagatgttgg ttcaacctct tttac 35
<210> 75
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 75
tattgtacaa agaagcacta catagtgcac ac 32
<210> 76
<211> 38
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 76
actatgtagt gcttctttgt acaataatga tagctatg 38
<210> 77
<211> 46
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 77
cataccaagg ccatttaata taagttttta aaatagaaag tgtttc 46
<210> 78
<211> 27646
<212> DNA
<213> avian infectious bronchitis Virus
<400> 78
actaaaaata gatattaata tatatctatt acactagcct tgcgctagat ttccaactta 60
acaaaacgga cttaaatacc tacagctggt cctcataggt gttccattgc agtgcacttt 120
agtgccctgg atggcacctg gccacctgtc aggtttttgt tattaaaatc ttattgttgc 180
tggtatcact gcttgttttg ccgtgtctca ctttatacat ccgttgcttg ggctacctag 240
tatccagcgt cctacgggcg ccgtggctgg ttcgagtgcg aagaacctct ggttcatcta 300
gcggtaggcg ggtgtgtgga agtagcgctt cagacgtact ggttctgttg cgtgaaacgc 360
ggggtcacct ccccccacat acctctaagg gcttttgagc ctagcgttgg gctacgttct 420
cgcacaaggt cggctatacg acgtttgtag ggggtagtgc caaacaaccc ctgaggtgac 480
aggttctggt ggtgtttagt gagcagacat acaatagaca gtgacaacat ggcttcaagc 540
ctaaaacagg gagtatctcc caaaccaagg gatgtcattc ttgtttccaa agacattccc 600
gaacaactct gtgacgcttt atttttctac acgtcacaca accctaagga ttacgctgat 660
gcttttgcat ttaggcaaaa gtttgaccgt aatctgcaga ctgggaagca gttcaaattt 720
gaaactgttt gtggtctctt cctattgaag ggagttgaca aaataacacc tggcgtccca 780
gcaaaagttt taaaagccac ttctaagttg gcagatttag aagacatctt tggtgtctct 840
ccttttgcac ggaagtaccg tgaattgttg aaaacagcat gccagtggtc tcttactgta 900
gaaacactgg atgctcgtgc acaaacgctt gacgaaattt ttgactctac tgaaatactt 960
tggcttcagg tggctgcaaa aattcaagtt tcagctatgg caatgcgcag gcttgttgga 1020
gaagtaactg caaaagtcat ggaagctctt ggctcaaatt tgagtgttct ctttcaaatt 1080
gttaaacaac aaatagccag aatctttcaa aaggcactgg ctatttttga aaatgtgagt 1140
gaattaccac agcgtattgc agcacttaag atggcctttg ccaagtgtgc caagtcaatt 1200
actgttgtgg ttgtggaaag aactctagtt gttagagagt tcgcaggaac ttgtcttgca 1260
agcatcaatg gtgctgttgc aaaattcttt gaagaacttc caaatggctt tatgggttct 1320
aaaatcttca caacattggc cttctttaaa gaagcagctg tgaaaattgt ggaaaatata 1380
ccaaatgcac caagaggtac tagaggtttt gaagtcgttg gtaacgccaa gggaacgcaa 1440
gttgttgtgc gtggcatgcg aaatgattta actctgctcg accaaaaagc tgacattcct 1500
gttgagaaag aaggttggtc tgcaattctt gaaggacatc tgtgttatgt ctttaagagt 1560
ggtgatcgtt tttatgcggc acctctttca gggaattttg cattgcatga tgtgcattgt 1620
tgtgagcgtg ttgtctgtct gtctgatggt gtaacaccag agataaatga tggactcatt 1680
ctagcagcaa tctattcatc ttttagtgtc tcagaactcg tggcagcact taaaaagggt 1740
gaaccattca agttcttggg tcataaattt gtgtatgcga aggatgcagc agtctctttc 1800
actcttgcaa aagcagccac tattgcagat gtactgaagc tgtttcaatc agctcgtgtg 1860
caaacagaag atgtgtggtc tgcatttact gaaaagtctt ttaatttctg gaaactcgca 1920
tatggaaaag tgcgtaatct tgaagaagtt gtgaagactc atttttgtaa agctcaaatg 1980
tcaattatca ttctagcagc agtgcttggc gaaggcattt ggcatcttgt ttcacaggtc 2040
atctataaag taggtggtct ttttactaga gtcgttgact tttgtgaaaa acactggaag 2100
ggtttctgtg cacaacttaa aaaggctaag ctcgttgtca cagaaactct ttgtgttctt 2160
aagggagtgg cacagcattg ttttcaacta ttgctggatg caatacattc tttgtatatg 2220
agttttaaga agtgtgcact tggtagaatt catggagact tactcttctg gaaagggggt 2280
gtacacaaaa ttgttcaaga tggcgatgaa gtttggtttg acgccattga tagtattgat 2340
gttgaagatc tgggtgttgt ccaagaaaaa cccatagatt ttgaggtttg tgaagacgta 2400
acacttccag aaaatcaacc tggtcatatg gttcaaatcg aggatgacgg aaagaactat 2460
atgttcttcc gcttcaaaag ggatgagaac atctactata caccaatgtc tcaacttggt 2520
gcaattaatg tagtttgcaa agcaggcggt aaaaccgtta cctttggaga caccattgtg 2580
aaagaaatac cgccacctga tgttgtgcct attaaggtta gcatagagtg ttgtggtgaa 2640
ccatggaata caatcttcaa gaaagcttat aaagagccca ttgaagttga aactgacctc 2700
acagtagaac aattgctctc tgtgatctat gagaaaatgt gcgacgacct caaattgttt 2760
ccagaggcac cagagccacc gccatttgag aatgtcgcac ttgttgataa aaacggtaaa 2820
gacttggatt gcataaaatc atgccatctt atctaccgtg attatgagag tgatgatgac 2880
atcgaggaag aagatgctga ggagtgtgat actgatttag aatgtgaaga agaggatgag 2940
gatactaaag tgttggctct tatacaagac cctgcaagta ataaataccc tcttcctctt 3000
gatgatgatt atagcgtctt taatggatgt attgtacata aggacgctct tgacgtcgta 3060
aatctaccat ctggtgaaga aacctttgtt gtcaacaact gctttgaggg agctgtaaaa 3120
ccactgcctc agaaagttgt tgatgttcta ggtgactggg gtgaggctgt tgatgcgcaa 3180
gagcaaattg cacaaactac ttcagaggaa acccctatca gtagtttgga ggcaactatt 3240
gagcaagttg ttgttgagga acagaaaata atttctgttg ttgaagaaga acagcaggtg 3300
gcggtctaca cacctgcaga cctacaagtt gttgaagaaa caccagatga gtttattctt 3360
attgctgatg tttccacaga agaaattgtg cctcatgaag aaaaggagtc acagattgaa 3420
caggagccta ttcaagttgt taaatcacaa cgtgaaaaga aggctaaaaa gttcaaggtt 3480
aaatctacta catgtgagaa acccaaattt ttggagtaca caacatgtgt gggtgaccta 3540
acggtagtga ttgccaaagc attggatgag tttaaagagt tctgcattgt aaatgctgct 3600
aatgagcata tgtctcacgg tggcggcgtt gcaaaggcaa ttgcagactt ttgtggaccg 3660
gactttgttg aatattgcgc ggactatgtt aagaaacatg gtccacagca aaaacttgtc 3720
acaccttcat ttgttaaagg cattcaatgt gtgaataatg ttgtaggacc tcgccatgga 3780
gacagcaact tgcgtgagaa gcttgttgct gcttacaaga gtgttcttgt aggtggagtg 3840
gttaactatg ttgtgccagt tctctcatca gggatttttg gtgtagattt taaaatgtca 3900
atagatgcta tgcgcgaagc ttttaaaggt tgtgccatac gcgttctttt attttctctg 3960
agtcaagaac acatcgatta tttcgatgca acttgtaagc agaagacaat ttatcttacg 4020
gaggatggtg ttaaataccg ctctgttgtt ttaaaacctg gtgattcttt gggtcaattt 4080
ggacaggttt ttgcaagaaa taaggtagtc ttttcggctg atgatgttga ggataaagaa 4140
atcctcttta tacccacaac tgacaagact attcttgaat attatggttt agatgcgcaa 4200
aagtatgtaa tatatttgca aacgcttgcg cagaaatggg atgttcaata tagagacaat 4260
tttgttatat tagagtggcg tgacggaaat tgctggatta gttcagcaat agtgctcctt 4320
caagctgcta agattaggtt taaaggtttt cttgcagaag catgggcaca acttttgggt 4380
ggagacccaa ctgattttgt agcctggtgc tatgcaagtt gcaatgctaa tgttggtgag 4440
ttttcagatg ctaattggct tcttgctaat ttggcagaat actttgatgc tgattacacg 4500
aatgcattcc ttaagaggcg tgtgtcatgt aactgtgggg ttaagaattg tgaagttaga 4560
ggccttgaag cttgtattca accagtaaag gcacccaatc ttcttcattt caagactcag 4620
tacacaaatt gtacagtgtg tgatgcaaat agtgtggatg aggtggtaga ggcctcacta 4680
ccatatctgt tgctccttgc tactgatggt cctactacag tggattgtga tgaaaatgct 4740
gtagggaatg ttgttttcat tggctctact aatagtggcc attgttacac gcaagccatt 4800
ggtaaggctt ttgataatct tgctaaggat agaaaatttt caaagaattc gccatacatt 4860
acagcaatgt atacgcgctt ctctcttaag agtgaaagct ctctgtctgt tgttaaacag 4920
agtaagagta aaactaaagt agtaaaagaa gatgttgcca accttgttac tagttctaaa 4980
gccagttttg atgatcttac tgactttgaa cattggtatg atagtaacat ctatgaaagt 5040
cttaaagttc aggaaatacc tgtgaatttg gatgagtatg tgtcatttac aacgaaagaa 5100
gatactaagt tgccactgac acttaaagtt agaggtatca aatcagttgt tgactttatt 5160
tcaagagacg gtttctctta taagttaaca cctgacattg aagaaaattc aaaagcgcca 5220
gtctactacc cagtcttaga ctctattagt cttaaggcaa tatgggtaga cggcagtgct 5280
aattttgttg ttggtcatcc aaactactat agtaagtctc ttcgcattcc tactttttgg 5340
gaaaatgcag agagctttgt taagataggt gacaaagttg atggtgtaac tatgggcctt 5400
tggcgtgcag aacagcttaa caaacctaat cttgaaagaa ttttcaacat tgctaagaaa 5460
gctattgttg gatccagtgt tgttactaca caatgtagta aattaattag taaagcagct 5520
acattcattg ctgataaagt aggtgggggt gtagttcgta atattacaga tagaattaag 5580
ggtctttgtg gatttacacg tgggcatttt gaaagaaaat tgtctccaca attcataaaa 5640
acacttatat tcttcttctt ttactttgta aaggctagtg ctaagagtgt tgccactagt 5700
tataagcgtg tgttatgtaa ggtggttttt accacgctat ttatattatg gtttatgtac 5760
acaagcaaac cagtaacttt tactggaaca cgtgtgctag acttcttatt tgagggttct 5820
ttatgtggtc cctataatga ctatggtaaa gactcatttg acgtactacg ctattgtgga 5880
gatgatttta cttgtcgtgt atgtttacat gataaagatt cacttcattt gtataagcat 5940
gcttatagcg tagaacaggt ttataaagat gcagcttctg gcattagttt taattggaat 6000
tggctttatt tggtctttct aatattattt gttaaaccag tagcaggttt cgttattatt 6060
tgctattgtg ttaagtactt ggtattgagt tcaactgtgt tgcaaactgg tgtaggtttt 6120
atggactggt ttattcaaac agtttttact cactttaatt ttatgggtgc aggtttctat 6180
ttctggctct tttataaatt gtacatacag gttcatcata tactgtattg taaggatata 6240
acatgtgaag tgtgtaagag agttgcacgc agtaacaggc atgaggttag tgttgttgtt 6300
ggtggacgca agcaaattgt gcatgtgtac actaactctg gttacaactt ttgtaagaga 6360
cataattggt attgtaggaa ttgtgatgta tatggtcacc aaaacacatt tatgtctcct 6420
gaagttgctg gcgagctttc tgaaaagctt aaacgccatg ttaaacctac agcacatgct 6480
taccacgttg tggatgaggc ttgcgtagtt gatgattttg ttaacttaaa atacaaagct 6540
gcaactcctg gtaaggatgg tgcacctcct gcagttaaat gtttcagtgt tacagatttc 6600
ttgaagaaag ctgtttttct taaggatgca ctgaaatgtg aacaaatatc taatgatggt 6660
tttatagtgt gtaatacgca gagtgcgcat gctttagagg aagcaaagaa tgcagccatc 6720
tattatgcgc aatacctgtg taaacctata cttatactcg accaggcact ctaccagaat 6780
ttaatagtgg aacctgtatc gaagagcgtt gtcaacaaag tgtgtgacat tttgtctagg 6840
ataatttctg tagatactgc atctttggat tataaagcag gtacaattcg tgatgccttg 6900
ctgtctgtta ctaaagatga agaagctgta gatatggcta tcttctgtca taatcatgaa 6960
gttgaatata caggtgatgg ttttactaat gttataccgt catatggtat agacactgat 7020
aaattaacac ctcgtgatag agggtttttg ataaatgcag atgcttctgt tgctaactta 7080
agagttaaaa atgctccgcc ggtagtatgg aagttctctg atcttattaa gttgtctgac 7140
agttgtctta aatatttaat ctcagcaact gtcaagtcag ggtctcgttt ctttataaca 7200
agatctggtg ctaaacaaat tttttcttgt agtactcaga aattgttggt agagaaaaag 7260
gctggtggtg tcattagtgg tacctttaat tggtttaaga gttgttgtaa atggctcttg 7320
atcttctatg tgctttttac attgtgttgt ttgggttgtt atcatatgga gacgaataaa 7380
agttttgttc atcctatgta tgatgttaac tctacaatgc atgttgaagg ctttaaggtt 7440
atagataaag gtgttattag agacattgta ccagaggatg cttgtttctc taataagttt 7500
gctaactttg atgcattttg gggtaaacca tatgtgaata gtagagactg tccaattgtt 7560
acagcagtca tagatggcgc tggaacaata gcagctggtg ttcctggttt tgtagactgg 7620
gttcttgatg gtgttatgtt tgtacacatg acacaaacag aaagaaaacc ctggtacatt 7680
cccacgtggt ttaacagaga aattgttggt tacactcagg attcaattat tactgaaggt 7740
agtttttata catctatagc tttgttttca gctaggtgtt tatatttaac agccagcaat 7800
acaccacaat tgtattgttt taatggtcat aatgatgctc ctggagcctt accatttagc 7860
agtatcactc cacacagggt ctacttccaa ccaaatggtg ttaggcttat aattcctcaa 7920
cagataatgc acacacccta cgtagtaaag tttttatcag acagctattg tagaggtagt 7980
gtatgtgagt atactaaacc gggttattgt gtctcactaa attcccaatg ggttttattt 8040
aatgacgaat acacaagtaa accaggagta ttctgtggtt ctactgttag agaacttatg 8100
tttaatatgg ttagtacatt ttttactggt gtcaacccta atatttatat gcagctggcg 8160
actatgttct taatactagt tgttgttgtg ttaatttttg caatggttat aaagtttcaa 8220
ggtgttttta aagcttatgc aaccattgtg tttacaataa tgctagtttg ggttgttaat 8280
gcatttattt tgtgtgtaca tagttataat agtgttgtgg ctgttatact actagtaatc 8340
tattgttatg catcattggt tacaagtcgt aatactgcta taataatgca ttgttggctt 8400
gtgtttacct ttggtttaat tgtacccata tggttggcgt gttgctacct ggcatttgtt 8460
ttatatatgt acacaccatt gtttttctgg tgttacggta ctactaaaaa cactcgtaaa 8520
ttgtatgatg gcaacgagtt tgttggtact tatgatcttg ctgcgaagag cacctttgtt 8580
attcgcggtc ctgaatttgt taagcttacg aacgagatag gtgataagtt tgaacactat 8640
ctctcagcgt atgctagact taaatactac tcaggcactg gcagtgaaca agattatctg 8700
caagcttgtc gtgcatggtt agcttatgct ttggaccaat atagaaatag tggtgtggaa 8760
attgtgtata ctccaccacg ttactctatt ggtgttagta gattacaggc cggttttaag 8820
aaactagttt ctcctagtag tgttgttgaa aagtgcattg ttagtgtctc ttatagaggt 8880
aataatctta atggactgtg gctaggtggt actatctact gtccgcgaca tgttctaggc 8940
aagttctcag gtgaccaatg gaatgatgta cttaaccttg ctaataatca tgagtttgaa 9000
gttgtaactc aaaataatgt tactttgaat gttgtcagta ggaggctaaa aggtgcagtt 9060
ttgattttac aaactgctgt tgctaatgct gaaactccaa agtataagtt tgttaaagcc 9120
aattgtggag atagttttac gattgcttgt tcctatggtg gtacagttgt tggactctac 9180
cctgttacta tgcgctctaa tggtactatt agggcgtctt tcttagcagg agcgtgtggt 9240
tctccaggtt ttaatataga aaagggtgta gttaactttt attatatgca ccatcttgag 9300
ttgcctaatg cattacacac aggaactgac ctaatgggag agttctatgg tggttatgtg 9360
gacgaagagg ttgcacaaag ggtgccacca gataatttag ttactaacaa tattgtagca 9420
tggctttatg ccgcaattat tagtgttaag gagagtagtt tctcactgcc taaatggttg 9480
gatagtacta ctgtcagtgt tgaagactgc aataagtggg ctggtgataa tggttttaca 9540
ccattttcta ctagtactgc tattactaaa ttaagtgcta taacaggagt agatgtttgt 9600
aaactccttc gcactattat ggtaaaaagt agtcaatggg gtagtgatcc cattttagga 9660
caatataatt ttgaagatga attgacacca gagtccgttt ttaatcagat aggtggtgtt 9720
agattacagt cttcttttgt aagaagagcc acatcctggt tttggagtag atgtgtgtta 9780
gcttgcttct tatttgtgtt gtgtgctatt gtcttgttta cggcagtgcc acttaaatat 9840
tatgtacatg cagccgttat tttgttaaca gctgtgctct tcatttcttt tactgttaaa 9900
catgttatgg catatatgga tacttttcta ctgccaacat tgcttacagt cattattgga 9960
gtttgtgctg aagtaccttt catctacaat actctaatta gtaggatagt tgtctttgtt 10020
agtcaatggt atgatcctgt agtctttgat actatggtac catggatgtt cttgccacta 10080
gtgttgtaca cagcatttaa gtgtgtgcag ggttgctata gtgtgaattc tttcaatact 10140
tctttgctag tactgtacca gttcttgaag ttaggctttg ttatttatgc ctcttctagc 10200
acgctggcag catacacaga aggtaattgg gatttatttt ttgaattagt tcacactact 10260
gtgttggcta atgttagtag taattcctta ataggtttgt ttgtgttcaa gttagctaag 10320
tggatgttgt attattgtaa tgctacatac tttaataatt atgtgctaat ggctgtcatt 10380
attaatggct ttggttggct cttcacttgt tactttggag tttattggtg gattaataag 10440
gtttttggtt taaccttagg taaatatgaa ttcaaagttt cagtagacca atataggtat 10500
atgtgtcttc ataagataaa ttcgcctaaa actgtgtggg aagttttttc gacaaatata 10560
cttatacaag gaattggtgg tgatcgtgtg ttgcctatag ctacagtgca atctaaattg 10620
agtgatgtaa agtgtacaac tgttgtttta atgcagcttt tgactaagct taatgttgaa 10680
gcaaattcaa aaatgcatgc ttatcttgtt gagttacaca ataaaatcct cgcatctgat 10740
gatgttggag agtgcatgga taatttattg ggtatgctta taacactatt ttgtatagat 10800
tctactattg atttgggtga gtattgtgat gatatactta agaggtcaac tgtattacaa 10860
tcggttactc aagagttttc gcacataccc tcgtatgctg aatatgaaag agctaagagt 10920
atttatgaaa aggttttagc cgattctaaa aatggtggtg taacacagca agagcttgct 10980
gcatatcgta aagctgccaa tattgcaaag tcagtttttg atagagactt ggctgttcaa 11040
aagaagttag atagcatggc agaacgtgct atgacaacaa tgtataaaga ggcgcgtgta 11100
actgatagaa gagcaaaatt agtttcatca ttacatgcac tacttttttc aatgcttaag 11160
aaaatagatt ctgagaagct taatgtctta tttgaccagg cgaatagtgg tgttgtaccc 11220
ctagcaactg ttccaattgt ctgtagtaat aagcttaccc tcgttatacc agacccagag 11280
acgtgggtca agtgtgtgga gggtgtgcat gttacatatt caacagttgt ttggaatata 11340
gactgtgtta ctgatgccga tggcacagag ttacacccca cttctacagg tagtggattg 11400
acttactgta taagtggtga taatatagca tggcctttaa aggttaactt gactaggaat 11460
gggcataata aggttgatgt tgccttgcaa aataatgagc ttatgcctca cggtgtaaag 11520
acaaaggctt gcgtagcagg tgtagatcaa gcacattgta gcgttgagtc taaatgttat 11580
tatacaagta ttagtggcag ttcagttgta gctgctatta cctcttcaaa tccaaatctg 11640
aaagtagcct cttttttgaa tgaggcaggt aatcagattt atgtagactt agacccacca 11700
tgtaaatttg gtatgaaagt gggtgataag gttgaagttg tttacctgta ttttataaaa 11760
aatacgaggt ctattgtaag aggtatggta cttggtgcta tatctaatgt tgttgtgtta 11820
caatctaaag gtcatgagac agaggaagtg gatgctgtag gcattctctc actttgttct 11880
tttgcagtag atcctgcgga tacatattgt aaatatgtgg cagcaggtaa tcaaccttta 11940
ggtaactgtg ttaaaatgtt gacagtacat aatggtagtg gttttgcaat aacatcaaag 12000
ccaagtccaa ctccggatca ggattcttat ggaggagctt ctgtgtgtct ttattgtaga 12060
gcacatatag cacaccctgg cggagcagga aatttagatg gacgctgtca atttaaaggt 12120
tcttttgtgc aaatacctac tacggagaaa gatcctgttg gattctgtct acgtaacaag 12180
gtttgcactg tttgtcagtg ttggattggt tatggatgtc agtgtgattc acttagacaa 12240
cctaaacctt ctgttcagtc agttgctgtt gcatctggtt ttgataagaa ttatttaaac 12300
gggtacgggg tagcagtgag gctcggctga taccccttgc taatggatgt gaccccgatg 12360
ttgtaaagcg agcctttgat gtttgtaata aggaatcagc cggtatgttt caaaatttga 12420
agcgtaactg tgcacgattc caagaagtac gtgatactga agatggaaat cttgagtatt 12480
gtgattctta ttttgtggtt aaacaaacca ctcctagtaa ttatgaacat gagaaagctt 12540
gttatgaaga cttaaagtca gaagtaacag ctgatcatga tttctttgtg ttcaataaga 12600
acatttataa tattagtagg cagaggctta ctaagtatac tatgatggat ttttgctatg 12660
ctttgcggca ctttgaccca aaggattgcg aagttcttaa agaaatactt gtcacttatg 12720
gttgtataga agattatcac cctaagtggt ttgaagagaa taaggattgg tacgacccaa 12780
tagaaaaccc taaatattat gccatgttgg ctaaaatggg acctattgta cgacgtgctt 12840
tattgaatgc tattgagttc ggaaacctca tggttgaaaa aggttatgtt ggtgttatta 12900
cacttgataa ccaagatctt aatggcaaat tttatgattt tggtgatttt cagaagacag 12960
cgcctggtgc tggtgttcct gtttttgata cgtattattc ttacatgatg cccatcatag 13020
ccatgactga tgcgttggca cctgagaggt attttgaata tgatgtgcat aagggttata 13080
aatcttatga tctcctcaag tatgattata ctgaggagaa acaagagttg tttcagaagt 13140
actttaagta ttgggatcaa gagtatcacc ctaactgtcg cgactgtagt gatgacaggt 13200
gtttgataca ttgtgcaaac ttcaacatct tgttttctac acttgtaccg cagacttctt 13260
tcggtaattt gtgtagaaag gtttttgttg atggtgtacc atttatagct acttgtggct 13320
atcattctaa ggaacttggt gttattatga atcaagataa caccatgtca ttttcaaaaa 13380
tgggtttgag tcaactcatg cagtttgttg gagatcctgc cttgttagtg gggacatcca 13440
ataaattagt ggatcttaga acgtcttgtt ttagtgtttg tgctttagcg tctggtatta 13500
ctcatcaaac ggtaaaacca ggtcacttta acaaggattt ctacgatttt gcagagaagg 13560
ctggtatgtt taaggaaggt tcttctatac cacttaaaca tttcttctac ccacagactg 13620
gtaatgctgc tataaacgat tatgattatt atcgttataa caggcctacc atgtttgata 13680
tacgtcaact tctattttgt ttagaagtga cttctaaata ttttgaatgt tatgaaggcg 13740
gctgtatacc agcaagccaa gttgtagtta acaatttaga taagagtgca ggttatccgt 13800
tcaataagtt tggaaaggcc cgtctctatt atgaaatgag tctagaggag caggaccaac 13860
tctttgagag tacaaagaag aacgtcctgc ctactataac tcagatgaat ttaaaatatg 13920
ccatatccgc gaaaaataga gcgcgtacag tggcaggtgt gtctatcctt tctactatga 13980
ctaataggca gtttcatcag aagattctta agtctatagt caacactaga aacgctcctg 14040
tagttattgg aacaaccaag ttttatggcg gttgggataa catgttgaga aaccttattc 14100
agggtgttga agacccgatt cttatgggtt gggattatcc aaagtgtgat agagcaatgc 14160
ctaatttgtt gcgtatagca gcatctttag tactcgctcg taaacacact aattgttgta 14220
cttggtctga acgcgtttat aggttgtata atgaatgcgc tcaggtttta tctgaaactg 14280
tcttagctac aggtggtata tatgtgaaac ctggtggtac tagcagtgga gatgctacta 14340
ctgcttatgc aaacagtgtt ttcaacataa tacaagccac atctgctaat gttgcgcgtc 14400
ttttgagtgt tataacgcgt gatattgtat atgatgacat taagagcttg cagtatgaat 14460
tgtaccagca ggtttatagg cgagtcaatt ttgacccagc atttgttgaa aagttttatt 14520
cttatttgtg taagaatttc tcattgatga tcttgtctga cgacggtgtt gtttgttata 14580
acaacacatt agccaaacaa ggtcttgtag cagatatttc tggttttaga gaagttctct 14640
actatcagaa caatgttttt atggctgatt ctaaatgttg ggttgaacca gatttagaaa 14700
aaggcccaca tgaattttgt tcacagcaca caatgttagt ggaggttgat ggtgagccta 14760
gatacttgcc atatccagac ccatcacgta ttttgtgtgc atgtgttttt gtagatgatt 14820
tggataagac agaatctgtg gctgttatgg agcgttatat cgctcttgcc atagatgcgt 14880
acccactagt acatcatgaa aatgaggagt acaagaaggt attctttgtg cttctttcat 14940
acatcagaaa actctatcaa gagctttctc agaatatgct tatggactac tcttttgtaa 15000
tggatataga caagggtagt aaattttggg agcaggagtt ctatgaaaat atgtatagag 15060
cccctacaac attacagtct tgtggcgttt gtgtagtgtg taatagtcaa actatattgc 15120
gctgtggtaa ttgtattcgc aaaccatttt tgtgttgtaa gtgttgctat gaccatgtca 15180
tgcacacaga ccacaaaaat gttttgtcta taaatcctta catttgctca cagccaggtt 15240
gtggtgaagc agatgttact aaattgtacc tcggaggtat gtcatacttc tgcggtaatc 15300
ataaaccaaa gttatcaata ccgttagtat ctaatggtac agtgtttgga atttacaggg 15360
ctaattgtgc aggtagcgaa aatgttgatg attttaatca actagctact actaattggt 15420
ctactgtgga accttatatt ttggcaaatc gttgtgtaga ttcgttgaga cgctttgctg 15480
cagagacagt aaaagctaca gaagaattac ataagcaaca atttgctagt gcagaagtga 15540
gagaagtact ctcagatcgt gaattgattc tgtcttggga gccaggtaaa accaggcctc 15600
cattgaatag aaattatgtt ttcactggct ttcactttac tagaactagt aaagttcagc 15660
tcggtgattt tacatttgaa aaaggtgaag gtaaggacgt tgtctattat cgagcgacgt 15720
ctactgctaa attgtctgtt ggagacattt ttgttttaac ctcacacaat gttgtttctc 15780
ttatagcgcc aacgttgtgt cctcagcaaa ccttttctag gtttgtgaat ttaagaccta 15840
atgtgatggt acctgcgtgt tttgtaaaca acattccatt gtaccattta gtaggcaagc 15900
agaagcgtac tacagtacaa ggccctcctg gcagtggtaa atcccatttt gctataggat 15960
tggcggctta ctttagtaac gcccgtgtcg tttttactgc atgctctcat gcagctgttg 16020
atgctttatg tgaagaagct tttaagtttc ttaaagtaga tgattgcact cgtatagtac 16080
ctcaaaggac tactatcgat tgcttctcta agtttaaagc taatgacaca ggcaaaaagt 16140
acatttttag tactattaat gccttgccag aagttagttg tgacattctt ttggttgacg 16200
aggttagtat gttgaccaat tacgaattgt cttttattaa tggtaagata aactatcaat 16260
atgttgtgta tgtaggtgat cctgctcaat taccggcgcc tcgtacgttg cttaacggtt 16320
cactctctcc aaaggattat aatgttgtca caaaccttat ggtttgtgtt aaacctgaca 16380
ttttccttgc aaagtgttac cgttgtccta aagaaattgt agatactgtt tctactcttg 16440
tatatgatgg aaagtttatt gcaaataacc cggaatcacg tcagtgtttc acggttatag 16500
ttaataatgg taattctgat gtaggacatg aaagtggctc agcctacaac acaactcaat 16560
tagaatttgt gaaagatttt gtctgtcgca ataaggaatg gcgggaagca acattcattt 16620
caccttataa tgctatgaac cagagagcct accgtatgct tggacttaat gttcagacag 16680
tagactcgtc tcaaggttcg gagtatgatt atgttatctt ttgtgttact gcagattcgc 16740
agcatgcact gaatattaac agattcaatg tagcgcttac aagagccaag cgtggtatac 16800
tagttgtcat gcgtcagcgt gatgaactat attcagctct taagtttata gagcttgata 16860
gtgtagcaag tctgcaaggt acaggcttgt ttaaaatttg caacaaagag tttagtggtg 16920
ttcatccagc ttatgcagtc acaactaagg ctcttgctgc aacttataaa gtaaatgatg 16980
aacttgctgc acttgttaac gtggaagctg gttcagaaat aacatataaa catcttattt 17040
ctttgttagg gtttaagatg agtgttaatg ttgaaggctg ccacaacatg tttataacac 17100
gtgatgaggc tatccgcaac gtaagaggtt gggtaggttt tgatgtagaa gcaacacatg 17160
cttgcggtac taacattggt actaacctgc ctttccaagt aggtttctct actggtgcag 17220
gctttgtagt tacgcctgag ggacttgtag atacttcaat aggcaataat tttgagcctg 17280
tgaattctaa agcacctcca ggtgaacaat ttaatcactt gagagcgtta ttcaaaagtg 17340
ctaaaccttg gcatgttgta aggccaagga ttgtgcaaat gttagcggat aacctatgca 17400
acgtttcaga ttgtgtagtg tttgtcacgt ggtgtcatgg cctagaacta accactttgc 17460
gctattttgt taaaataggc aaggaccaag tttgttcttg cggttctaga gcaacaactt 17520
ttaattctta tactcaggct tatgcttgtt ggaagcattg cttgggtttt gattttgttt 17580
ataatccact cttagtggat attcagcagt ggggttattc tggtaaccta caatttaacc 17640
atgatttgca ttgtaatgtg catggacacg cacatgtagc ttctgcggat gctattatga 17700
cgcgttgtct tgcaattaat aatgcatttt gtcaagatgt caactgggat ttaacttacc 17760
ctcatatagc aaatgaggat gaagtcaatt ctagctgtag atatttacaa cgcatgtatc 17820
ttaatgcatg tgttgatgct cttaaagtta acgttgtcta tgatataggc aaccctaaag 17880
gtataaaatg tgttagacgt ggagacttaa attttagatt ctatgataag aatccaatag 17940
tacccaatgt caagcagttt gagtatgact ataatcagca caaagataag tttgctgatg 18000
gtctttgtat gttttggaat tgtaatgtgg attgttatcc cgacaattcc ttagtttgta 18060
ggtacgacac acgaaatttg agtgtgttta acctacctgg ttgtaatggt ggtagcttgt 18120
atgtcaacaa gcatgcattc cacacaccta aatttgatcg cactagcttt cgtaatttga 18180
aagctatgcc attctttttc tatgactcat cgccttgcga gaccattcaa gtggatggag 18240
ttgcgcaaga ccttgtgtca ttagctacga aagattgtat cacaaaatgc aacataggcg 18300
gtgctgtttg taaaaagcac gcacaaatgt atgcagattt tgtgacttct tataatgcag 18360
ctgttactgc tggttttact ttttgggtta ctaataattt taacccatat aatttgtgga 18420
aaagtttttc agctctccag tctatcgaca atattgctta taatatgtat aagggtggtc 18480
attatgatgc tattgcagga gaaatgccca ctatcgtaac tggagataaa gtttttgtta 18540
tagatcaagg cgtagaaaaa gcagtttttt ttaatcaaac aattctgcct acatctgtag 18600
cgtttgagct gtatgcgaag agaaatattc gcacactgcc aaacaaccgt attttgaaag 18660
gtttgggtgt agatgtgact aatggatttg taatttggga ttacacgaac caaacaccac 18720
tataccgtaa tactgttaag gtatgtgcat atacagacat agaaccaaat ggcctaatag 18780
tgctgtatga tgatagatat ggtgattacc agtcttttct ggctgctgat aatgctgttt 18840
tagtttctac acagtgttac aagcggtatt cgtatgtaga aataccgtca aacctgcttg 18900
ttcagaacgg tattccgtta aaagatggag cgaacctgta tgtttataag cgtgttaatg 18960
gtgcgtttgt tacgctacct aacacattaa acacacaggg tcgcagttat gaaacttttg 19020
aacctcgtag tgatgttgag cgtgattttc tcgacatgtc tgaggagagt tttgtagaaa 19080
agtatggtaa agaattaggt ctacagcaca tactgtatgg tgaagttgat aagccccaat 19140
taggtggttt acacactgtt ataggtatgt gcagactttt acgtgcgaat aagttgaacg 19200
caaagtctgt tactaattct gattctgatg tcatgcaaaa ttattttgta ttggcagaca 19260
atggttccta caagcaagtg tgtactgttg tggatttgct gcttgatgat ttcttagaac 19320
ttcttaggaa catactgaaa gagtatggta ctaataagtc taaagttgta acagtgtcaa 19380
ttgattacca tagcataaat tttatgactt ggtttgaaga tggcattatt aaaacatgtt 19440
atccacagct tcaatcagca tggacgtgtg gttataatat gcctgaactt tacaaagttc 19500
agaattgtgt tatggaacct tgcaacattc ctaattatgg tgttggaata gcgttgccaa 19560
gtggtattat gatgaatgtg gcaaagtata cacaactctg tcaatacctt tcgaaaacaa 19620
caatgtgtgt accgcatagt atgcgagtaa tgcattttgg agctggaagt gacaaaggag 19680
tggctccagg tagtactgtt cttaaacaat ggctcccaga agggacactc cttgtcgata 19740
atgatattgt agactatgtg tctgatgcac atgtttctgt gctttcagat tgcaataaat 19800
ataagacaga gcacaagttt gatcttgtga tatctgatat gtatacagac aatgattcaa 19860
aaagaaagca tgaaggcgtg atagccaata atggcaatga tgacgttttc atatatctct 19920
caagttttct acgcaataat ttggctctgg gaggcagttt tgctgtaaaa ttaacagaga 19980
caagttggca tgagagttta tatgacattg cacaggattg tgcatggtgg acaatgttct 20040
gtacagcagt gaatgcatct tcttcagaag cattcctgat tggtgttaat tacttgggtg 20100
caagtgcaaa ggttaaagtt agtggaaaaa cactgcacgc aaattatata ttttggagga 20160
attgtaatta tttacaaacc tctgcttata gtatatttga tgttgctaag tttgatttga 20220
gattgaaagc aacgccagtt gttaatttga aaactgaaca aaagacagac ttagtcttta 20280
atttaattaa gtgtggtaag ttactggtaa gagatgttgg taacacctct tttactagtg 20340
actcttttgt gtgcactatg tagtgctgtt ttgtatgaca gtagttctta cgtgtactac 20400
taccaaagtg ccttcagacc acctgatggt tggcatttac atgggggtgc gtatgcggtt 20460
gttaatattt ctagtgaatc taataatgca ggctcttcat ctgggtgtac tgttggtatt 20520
attcatggtg gtcgtgttgt taatgcttct tctatagcta tgacggcacc gtcatcaggt 20580
atggcttggt ctagcagtca gttttgtact gcatactgta acttttcaga tactacagtg 20640
tttgttacac attgttataa acatgttggg tgtcctataa ctggcatgct tcaacagcat 20700
tctatacgtg tttctgctat gaaaaatggc cagccttttt ataatttaac agttagtgta 20760
gctaagtacc ctacttttaa atcatttcag tgtgttaata atttaacatc cgtatattta 20820
aatggtgatc ttgtttacac ctctaatgag accacagatg ttacatctgc aggtgtttat 20880
tttaaagctg gtggacctat aacttataaa gttatgagag aagttagagc cctggcttat 20940
tttgttaatg gtactgcaca agatgttatt ttgtgtgatg ggccacctag aggcttgtta 21000
gcatgccagt ataatactgg caatttttca gatggctttt atccttttac taatagtagt 21060
ttagttaagc agaagtttat tgtctatcgt gaaaatagtg ttaatactac ttttacgtta 21120
cgcaatttca cttttcataa tgagactggc gccaacccta atcctagtgg tgttcagaat 21180
attcaaactt accaaacaca aacagctcag agtggttatt ataattttaa tttttccttt 21240
ctgagtagtt ttgtttataa ggagtctaat tttatgtatg gatcttatca cccaagttgt 21300
aattttagac tagaaactat taataatggt ttgtggttta attcactttc agtttcaatt 21360
gcttacggtc ctcttcaagg tggttgcaag caatctgtct ttagtggtag agcaacctgt 21420
tgttatgctt actcatatgg aggtcctttg ctgtgtaaag gtgtttattc aggtgagtta 21480
ggtcataatt ttgaatgtgg actgttagtt tatgttacta agagcggtgg ctctcgtata 21540
caaacagcca ctgaaccgcc agttataact caacacaatt ataataatat tactttaaat 21600
acttgtgttg attataatat atatggcaga actggccaag gttttattac taatgtaacc 21660
gactcagctg ttagttataa ttatctagca gacgcaggtt tggctatttt agatacatct 21720
ggttccatag acatctttgt tgtacaaggt gaatatggtc ttacttatta taaggttaac 21780
ccttgcgaag atgtcaacca gcagtttgta gtttctggtg gtaaattagt aggtattctt 21840
acttcacgta atgagactgg ttctcagctt cttgagaacc agttttacat taaaatcact 21900
aatggaacac gtcgttttag acgttctatt actgaaaatg ttgaaaattg cccttatgtt 21960
agttatggta agttttgtat aaaacctgat ggttcaattg ccacaatagt accaaaacaa 22020
ttggaacagt ttgtggcacc tttacttaat gttactgaaa atgtgctcat acctaacagt 22080
tttaatttaa ctgttacaga tgagtacata caaacgcgta tggataaggt ccaaattaat 22140
tgtctgcagt atgtttgtgg caattctctg gattgtagag atttgtttcg acaatatggg 22200
catgtttgtg acaacatatt gtctgtagta aatagtattg gtcaaaaaga agatatggaa 22260
cttttgaatt tctattcttc tactaaaccg gttggtttta atacaccatt tcttagtaat 22320
gttagcactg gtgagtttaa tatttctctt ctgttaacaa ctcctagtag tcctagaagg 22380
cgttctttta ttgaagacct tctatttaca agcgttgaat ctgttggatt accaacagat 22440
gacgcataca aaaattgcac tgcaggacct ttaggttttc ttaaggacct tgcgtgtgct 22500
cgtgaatata atggtttgct tgtgttgcct cccattataa cagcagaaat gcaaactttg 22560
tatactagtt ctctagtagc ttctatggct tttggtggta ttactgcagc tggtgctata 22620
ccttttgcca cacaactgca ggctagaatt aatcacttgg gtattaccca gtcacttttg 22680
ttgaagaatc aagaaaaaat tgctgcttcc tttaataagg ccattggtcg tatgcaggaa 22740
ggttttagaa gtacatcttt agcattacaa caaattcaag atgttgttaa taagcagagt 22800
gctattctta ctgagactat ggcatcactt aataaaaatt ttggtgccat ttcttctgtg 22860
attcaagaaa tctaccagca acttgacgcc atacaagcaa atgctcaagt ggatcgtctt 22920
ataactggta gattgtcatc actttctgtt ttagcatctg ctaagcaggc ggagtatatt 22980
agagtgtcac aacagcgtga gttagctact cagaagatta atgagtgtgt taagtcacag 23040
tccattaggt actccttttg tggtaatgga cgacatgttt taaccatacc gcaaaatgca 23100
cctaatggta tagtgtttat acacttttct tacactccag atagttttgt taatgttact 23160
gcaatagtgg gtttttgtgt aaagccagct aatgctagtc agtatgcaat agtacccgct 23220
aatggtaggg gtattttcat acaagttaat ggtagttact acatcactgc acgagatatg 23280
tatatgccaa gagctattac tgcaggagat atagttacgc ttacttcttg tcaagtaaat 23340
tatgtaagtg taaataagac cgtcattact acattcgtag acaatgatga ttttgatttt 23400
aatgacgaat tgtcaaaatg gtggaatgat actaagcatg agctaccaga ctttgacaaa 23460
ttcaattaca cagtacctat acttgacatt gatagtgaaa ttgatcgtat tcaaggcgtt 23520
atacagggtc ttaatgactc tctaatagac cttgaaaaac tttcaatact caaaacttat 23580
attaagtggc cttggtatgt gtggttagcc atagcttttg ccactattat cttcatctta 23640
atattaggat gggttttctt catgactggg tgttgtggtt gttgttgtgg atgctttggc 23700
attatgcctc taatgagtaa gtgtggtaag aaatcttctt attacacgac ttttgataac 23760
gatgtggtaa ctgaacaata cagacctaaa aagtctgttt aatgatccaa agtcccacgt 23820
ccttcttaat agtattaatt ttgctttggt gtaaacttgt actaagttgt tttagagagt 23880
ttattatagc gcttcaacaa ctaacacaag ttttactcca aattatcgat agtaatttac 23940
agtctagact gaccctttgt cacagtctag actaatgtta aacttagaag caattattga 24000
aactggtgag caagtgattc aaaaaatcag tttcaattta cagcatattt caagtgtatt 24060
aaacacagaa gtatttgacc cctttgacta ttgttattac agaggaggta atttttggga 24120
aatagagtca gctgaagatt gttcaggtga tgatgaattt attgaataag tcgctagagg 24180
aaaatggaaa ttttctaaca gcgctttaca tatttgtagg atttttagca ctttatcttc 24240
taggtagagc acttcaagca tttgtacagg ctgctgatgc ttgttgttta ttttggtata 24300
catgggtagt aattccagga gctaagggta cagcctttgt atacaagtat acatatggta 24360
gaaaacttaa caatccggaa ttagaagcag ttattgtcaa cgagtttcct aagaacggtt 24420
ggaataataa aaatccagca aattttcaag atgtccaacg agacaaattg tactcttgac 24480
tttgaacagt cagttgagct ttttaaagag tataatttat ttataactgc attcttgttg 24540
ttcttaacca taatacttca gtatggctat gcaacaagag gtaagtttat ttatatactg 24600
aaaatgatag tgttatggtg cttttggccc cttaacattg cagtaggtgt aatttcatgt 24660
atatacccac caaacacagg aggtcttgtc gcagcgataa tacttacagt gtttgcgtgt 24720
ctgtcttttg taggttattg gatccagagt attagactct ttaagcggtg taggtcatgg 24780
tggtcattta acccagaatc taatgccgta ggttcaatac tcctaactaa tggtcaacaa 24840
tgtaattttg ctatagagag tgtgccaatg gtgctttctc caattataaa gaatggtgtt 24900
ctttattgtg agggtcagtg gcttgctaag tgtgaaccag accacttgcc taaagatata 24960
tttgtttgta caccggatag acgtaatatc taccgtatgg tgcagaaata tactggtgac 25020
caaagcggaa ataagaaaag gtttgctacg tttgtctatg caaagcagtc agtagacact 25080
ggcgagctag aaagtgtagc aacaggtgga agtagccttt acacataaat gtgtgtgtgt 25140
agagagtatt taaaattatt cttcaatagt gcctctattt taagagcgcg gaagagtatt 25200
tgttttgagg atattaatat aaatcctctt tgttttgtac tctctttaca agagttatta 25260
tttaagcaac agtttttcct ttcctttgtt tggaagaaag ttgttgttaa tggtgtagaa 25320
ttccaagtag aaaatggaaa agtccactac gaaggaaacc ccattttcta aaaaggttgt 25380
tgtaggttgt ggtcccatta taagaaggat taaatggatt aaaccaccta cactacttac 25440
ttgtaataag ggcgtttgga cttacaagcg cttaacaaat acagacgatg aaatggctga 25500
ctagttttgg aagagcagtt atttcttgtt ataaagccct actattaact cagttaagag 25560
tattagatag gttaatttta gatcacggac caaaacgcgt cttaacgtgt ggtaggcgag 25620
tgcttttatc tcaattagat ctagtttata ggttggcata tacgcccacc caatcgctgg 25680
tatgaataat agtaaagata atccttttcg cggagcaata gcaagaaaag cgcgaattta 25740
tctgagagaa ggattagagt gtgtttactt tcttaacaaa gcaggacaag cagagtcttg 25800
tcccgcgtgt acctctctag tattccaggg gaaaacttgt gaggaacaca aagataataa 25860
taatcttttg tcatggcaag cggtaaggca actggaaaga cagatgcccc aactccagtc 25920
atcaaactag gaggaccaaa gccacctaaa gttggttctt ctggaaatgt atcttggttt 25980
caagcaataa aagccaagaa gttaaattca cctccgccta agtttgaagg tagcggtgtt 26040
cctgataatg aaaatctaaa accaagtcag cagcatggat attggagacg ccaagctagg 26100
tttaagccag gtaaaggtgg aagaaaacca gtcccagatg cttggtattt ttactatact 26160
ggaacaggac cagccgctaa cctgaattgg ggtgatagcc aagatggtat agtgtgggtt 26220
gctggtaagg gtgctgatac taaatttaga tctaatcagg gtactcgtga ctctgacaag 26280
tttgaccaat atccgctacg gttttcagac ggaggacctg atggtaattt ccgttgggat 26340
ttcattcctc tgaatcgtgg taggagtgga agatcaacag cggcttcatc agcagcatct 26400
agtagagcac cgtcgcgtga tggctcgcgt ggacgtagaa gcggagctga agatgatctt 26460
atagctcgtg cagcaaagat cattcaggat cagcagaaga agggttctcg cattactaaa 26520
gctaaggccg atgaaatggc tcatcgccgg tattgtaagc gtactatccc acctggttat 26580
aaggttgatc aagtatttgg tccccgtact aaatgtaagg agggaaattt tggtgatgac 26640
aagatgaatg aggagggtat taaggatggg cgcgttacag caatgctcaa cctagtccct 26700
agcagccatg cttgtctttt tggaagtaga gtgacgccca aacttcaacc agatgggctg 26760
cacttgagat ttgaatttac tactgtggtt tcacgtgatg atccgcagtt tgataattat 26820
gtgaaaattt gtgatcagtg tgtcgatggt gtagggacgc gtccaaaaga cgatgaaccg 26880
agaccaaagt cacgcccaaa ttcaagacct gctacaagaa caagttctcc agcgccaaga 26940
caacagcgtc aaaagaagga gaagaagtca aagaagcagg atgatgaagt agataaggca 27000
ttgacctcag atgaggagag gaacaatgca cagctggaat ttgatgatga acccaaggtg 27060
attaactggg gggagtcagc acttggagag aatgagttgt aaagctagat ttccaactta 27120
acatcatgga cgtgcgtatg ctgtttttcc ctactataga ctttttagca tattattttt 27180
tgctatttgt atggtttatt acaggtgaag attgtatgta tttgttgtac actcgtatgt 27240
tctatattat gttttctgta gttgttatta gtgttgttct tgttcttact ctactgttct 27300
cttttcttta ttttagagta tcaataagaa tcaaggaaga taggcatgta gtttgattac 27360
ctacatgtct atcgccaggg aaatgtctaa tctgtctact tagtagcctg gaaacgaacg 27420
gtagaccctt agattttaat ttagtttaat ttttagttta gtttaagtta gtttagagta 27480
ggtataaaga agccagtgcc ggggccacgc ggagtacgat cgagggtaca gcactaggac 27540
gcccattagg ggaagagcta aattttagtt taagttaagt ttaataggct atgtatagtt 27600
aaaatttata ggctagtata gagttagagc aaaaaaaaaa aaaaaa 27646
<210> 79
<211> 1162
<212> PRT
<213> avian infectious bronchitis Virus
<400> 79
Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Ala Leu Cys
1 5 10 15
Ser Ala Val Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gln Ser
20 25 30
Ala Phe Arg Pro Pro Asp Gly Trp His Leu His Gly Gly Ala Tyr Ala
35 40 45
Val Val Asn Ile Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Ser Gly
50 55 60
Cys Thr Val Gly Ile Ile His Gly Gly Arg Val Val Asn Ala Ser Ser
65 70 75 80
Ile Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gln
85 90 95
Phe Cys Thr Ala Tyr Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr
100 105 110
His Cys Tyr Lys His Val Gly Cys Ser Leu Thr Gly Met Leu Gln Gln
115 120 125
His Ser Ile Arg Val Ser Ala Met Lys Asn Gly Gln Leu Phe Tyr Asn
130 135 140
Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gln Cys
145 150 155 160
Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr
165 170 175
Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala
180 185 190
Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Glu Val Arg Ala Leu Ala
195 200 205
Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys Asp Gly Ser
210 215 220
Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp
225 230 235 240
Gly Phe Tyr Pro Phe Thr Asn Ser Ser Leu Val Lys Gln Lys Phe Ile
245 250 255
Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe
260 265 270
Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gln
275 280 285
Asn Ile Gln Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn
290 295 300
Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe
305 310 315 320
Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile
325 330 335
Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala Tyr Gly
340 345 350
Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly Arg Ala Thr
355 360 365
Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Leu Leu Cys Lys Gly Val
370 375 380
Tyr Ser Gly Glu Leu Asp His Asn Phe Glu Cys Gly Leu Leu Val Tyr
385 390 395 400
Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro
405 410 415
Val Ile Thr Gln His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val
420 425 430
Asp Tyr Asn Ile Tyr Gly Gly Ile Gly Gln Gly Phe Ile Thr Asn Val
435 440 445
Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala
450 455 460
Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Phe Val Val Gln Ser Glu
465 470 475 480
Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gln
485 490 495
Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg
500 505 510
Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile
515 520 525
Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Ile Thr Glu Ser Val Glu
530 535 540
Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly
545 550 555 560
Ser Ile Ala Thr Ile Val Pro Lys Gln Leu Glu Gln Phe Val Ala Pro
565 570 575
Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu
580 585 590
Thr Val Thr Asp Glu Tyr Ile Gln Thr Leu Met Asp Lys Val Gln Ile
595 600 605
Asn Cys Leu Gln Tyr Ile Cys Gly Asn Ser Leu Glu Cys Arg Asn Leu
610 615 620
Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn
625 630 635 640
Ser Val Gly Gln Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser
645 650 655
Thr Lys Pro Ala Gly Phe Asn Thr Pro Val Leu Ser Asn Val Ser Thr
660 665 670
Gly Glu Phe Asn Ile Ser Leu Phe Leu Thr Thr Pro Ser Ser Pro Arg
675 680 685
Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val
690 695 700
Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu
705 710 715 720
Gly Phe Leu Lys Asp Leu Val Cys Ala Arg Glu Tyr Asn Gly Leu Leu
725 730 735
Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser
740 745 750
Ser Leu Val Ala Ser Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala
755 760 765
Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile
770 775 780
Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe
785 790 795 800
Asn Lys Ala Ile Gly His Met Gln Glu Gly Phe Arg Ser Thr Ser Leu
805 810 815
Ala Leu Gln Gln Ile Gln Asp Val Val Asn Lys Gln Ser Ala Ile Leu
820 825 830
Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser
835 840 845
Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala
850 855 860
Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Leu
865 870 875 880
Ala Ser Ala Lys Gln Ala Glu Tyr Ile Arg Val Ser Gln Gln Arg Glu
885 890 895
Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Arg
900 905 910
Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn
915 920 925
Ala Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Asp Ser
930 935 940
Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn
945 950 955 960
Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gly Arg Gly Ile Phe Ile
965 970 975
Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro
980 985 990
Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Leu Thr Ser Cys Gln Val
995 1000 1005
Asn Tyr Val Ser Val Asn Lys Thr Val Ile Thr Thr Phe Val Asp
1010 1015 1020
Asn Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn
1025 1030 1035
Asp Thr Lys His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr
1040 1045 1050
Val Pro Ile Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gln Gly
1055 1060 1065
Val Ile Gln Gly Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Leu
1070 1075 1080
Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu
1085 1090 1095
Ala Ile Ala Phe Ala Thr Ile Ile Phe Ile Leu Ile Leu Gly Trp
1100 1105 1110
Val Phe Phe Met Thr Gly Cys Cys Gly Cys Cys Cys Gly Cys Phe
1115 1120 1125
Gly Ile Met Pro Leu Met Ser Lys Cys Gly Lys Lys Ser Ser Tyr
1130 1135 1140
Tyr Thr Thr Phe Asp Asn Asp Val Val Thr Glu Gln Tyr Arg Pro
1145 1150 1155
Lys Lys Ser Val
1160
<210> 80
<211> 409
<212> PRT
<213> avian infectious bronchitis Virus
<400> 80
Met Ala Ser Gly Lys Ala Thr Gly Lys Thr Asp Ala Pro Thr Pro Val
1 5 10 15
Ile Lys Leu Gly Gly Pro Lys Pro Pro Lys Val Gly Ser Ser Gly Asn
20 25 30
Val Ser Trp Phe Gln Ala Ile Lys Ala Lys Lys Leu Asn Ser Pro Pro
35 40 45
Pro Lys Phe Glu Gly Ser Gly Val Pro Asp Asn Glu Asn Leu Lys Pro
50 55 60
Ser Gln Gln His Gly Tyr Trp Arg Arg Gln Ala Arg Phe Lys Pro Gly
65 70 75 80
Lys Gly Gly Arg Lys Pro Val Pro Asp Ala Trp Tyr Phe Tyr Tyr Thr
85 90 95
Gly Thr Gly Pro Ala Ala Asn Leu Asn Trp Gly Asp Ser Gln Asp Gly
100 105 110
Ile Val Trp Val Ala Gly Lys Gly Ala Asp Thr Lys Phe Arg Ser Asn
115 120 125
Gln Gly Thr Arg Asp Ser Asp Lys Phe Asp Gln Tyr Pro Leu Arg Phe
130 135 140
Ser Asp Gly Gly Pro Asp Gly Asn Phe Arg Trp Asp Phe Ile Pro Leu
145 150 155 160
Asn Arg Gly Arg Ser Gly Arg Ser Thr Ala Ala Ser Ser Ala Ala Ser
165 170 175
Ser Arg Ala Pro Ser Arg Glu Val Ser Arg Gly Arg Arg Ser Gly Ser
180 185 190
Glu Asp Asp Leu Ile Ala Arg Ala Ala Arg Ile Ile Gln Asp Gln Gln
195 200 205
Lys Lys Gly Ser Arg Ile Thr Lys Ala Lys Ala Asp Glu Met Ala His
210 215 220
Arg Arg Tyr Cys Lys Arg Thr Ile Pro Pro Asn Tyr Lys Val Asp Gln
225 230 235 240
Val Phe Gly Pro Arg Thr Lys Gly Lys Glu Gly Asn Leu Gly Asp Asp
245 250 255
Lys Met Asn Glu Glu Gly Ile Lys Asp Gly Arg Val Thr Ala Met Leu
260 265 270
Asn Leu Val Pro Ser Ser His Ala Cys Leu Phe Gly Ser Arg Val Thr
275 280 285
Pro Arg Leu Gln Pro Asp Gly Leu His Leu Lys Phe Glu Phe Thr Thr
290 295 300
Val Val Pro Arg Asp Asp Pro Gln Phe Asp Asn Tyr Val Lys Ile Cys
305 310 315 320
Asp Gln Cys Val Asp Gly Val Gly Thr Arg Pro Lys Asp Asp Glu Pro
325 330 335
Arg Pro Lys Ser Arg Ser Ser Ser Arg Pro Ala Thr Arg Gly Asn Ser
340 345 350
Pro Ala Pro Arg Gln Gln Arg Pro Lys Lys Glu Lys Lys Pro Lys Lys
355 360 365
Gln Asp Asp Glu Val Asp Lys Ala Leu Thr Ser Asp Glu Glu Arg Asn
370 375 380
Asn Ala Gln Leu Glu Phe Asp Asp Glu Pro Lys Val Ile Asn Trp Gly
385 390 395 400
Asp Ser Ala Leu Gly Glu Asn Glu Leu
405
<210> 81
<211> 409
<212> PRT
<213> avian infectious bronchitis Virus
<400> 81
Met Ala Ser Gly Lys Ala Ala Gly Lys Thr Asp Ala Pro Thr Pro Val
1 5 10 15
Ile Lys Leu Gly Gly Pro Lys Pro Pro Lys Val Gly Ser Ser Gly Asn
20 25 30
Val Ser Trp Phe Gln Ala Ile Lys Ala Lys Lys Leu Asn Ser Pro Pro
35 40 45
Pro Lys Phe Glu Gly Ser Gly Val Pro Asp Asn Glu Asn Leu Lys Pro
50 55 60
Ser Gln Gln His Gly Tyr Trp Arg Arg Gln Ala Arg Phe Lys Pro Gly
65 70 75 80
Lys Gly Gly Arg Lys Pro Val Pro Asp Ala Trp Tyr Phe Tyr Tyr Thr
85 90 95
Gly Thr Gly Pro Ala Ala Asn Leu Asn Trp Gly Asp Ser Gln Asp Gly
100 105 110
Ile Val Trp Val Ala Gly Lys Gly Ala Asp Thr Lys Phe Arg Ser Asn
115 120 125
Gln Gly Thr Arg Asp Ser Asp Lys Phe Asp Gln Tyr Pro Leu Arg Phe
130 135 140
Ser Asp Gly Gly Pro Asp Gly Asn Phe Arg Trp Asp Phe Ile Pro Leu
145 150 155 160
Asn Arg Gly Arg Ser Gly Arg Ser Thr Ala Ala Ser Ser Ala Ala Ser
165 170 175
Ser Arg Ala Pro Ser Arg Glu Val Ser Arg Gly Arg Arg Ser Gly Ser
180 185 190
Glu Asp Asp Leu Ile Ala Arg Ala Ala Arg Ile Ile Gln Asp Gln Gln
195 200 205
Lys Lys Gly Ser Arg Ile Thr Lys Ala Lys Ala Asp Glu Met Ala His
210 215 220
Arg Arg Tyr Cys Lys Arg Thr Ile Pro Pro Asn Tyr Lys Val Asp Gln
225 230 235 240
Val Phe Gly Pro Arg Thr Lys Gly Lys Glu Gly Asn Phe Gly Asp Asp
245 250 255
Lys Met Asn Glu Glu Gly Ile Lys Asp Gly Arg Val Thr Ala Met Leu
260 265 270
Asn Leu Val Pro Ser Ser His Ala Cys Leu Phe Gly Ser Arg Val Thr
275 280 285
Pro Arg Leu Gln Pro Asp Gly Leu His Leu Lys Phe Glu Phe Thr Thr
290 295 300
Val Val Pro Arg Asp Asp Pro Gln Phe Asp Asn Tyr Val Lys Ile Cys
305 310 315 320
Asp Gln Cys Val Asp Gly Val Gly Thr Arg Pro Lys Asp Asp Glu Pro
325 330 335
Arg Pro Lys Ser Arg Ser Ser Ser Arg Pro Ala Thr Arg Gly Asn Ser
340 345 350
Pro Ala Pro Arg Gln Gln Arg Pro Lys Lys Glu Lys Lys Pro Lys Lys
355 360 365
Gln Asp Asp Glu Val Asp Lys Ala Leu Thr Ser Asp Glu Glu Arg Asn
370 375 380
Asn Ala Gln Leu Glu Phe Asp Asp Glu Pro Lys Val Ile Asn Trp Gly
385 390 395 400
Asp Ser Ala Leu Gly Glu Asn Glu Leu
405
<210> 82
<211> 109
<212> PRT
<213> avian infectious bronchitis Virus
<400> 82
Met Met Asn Leu Leu Asn Lys Ser Leu Glu Glu Asn Gly Ser Phe Leu
1 5 10 15
Thr Ala Leu Tyr Ile Phe Val Gly Phe Leu Ala Phe Tyr Leu Leu Gly
20 25 30
Arg Ala Leu Gln Ala Phe Val Gln Ala Ala Asp Ala Cys Cys Leu Phe
35 40 45
Trp Tyr Thr Trp Leu Val Ile Pro Gly Val Lys Gly Thr Ala Phe Val
50 55 60
Tyr Lys Tyr Thr Tyr Gly Arg Lys Leu Asn Asn Ser Glu Leu Glu Ala
65 70 75 80
Val Val Val Asn Glu Phe Pro Lys Asn Gly Trp Asn Asn Lys Asn Pro
85 90 95
Ala Asn Phe Gln Asp Val Gln Arg Asn Lys Leu Tyr Ser
100 105
<210> 83
<211> 225
<212> PRT
<213> avian infectious bronchitis Virus
<400> 83
Met Ser Asn Glu Thr Asn Cys Thr Leu Asp Phe Glu Gln Ser Val Glu
1 5 10 15
Leu Phe Lys Glu Tyr Asn Leu Phe Ile Thr Ala Phe Leu Leu Phe Leu
20 25 30
Thr Ile Ile Leu Gln Tyr Gly Tyr Ala Thr Arg Ser Lys Phe Ile Tyr
35 40 45
Ile Leu Lys Met Ile Val Leu Trp Cys Phe Trp Pro Leu Asn Ile Ala
50 55 60
Val Gly Val Ile Ser Cys Ile Tyr Pro Pro Asn Thr Gly Gly Leu Val
65 70 75 80
Ala Ala Ile Ile Leu Thr Val Phe Ala Cys Leu Ser Phe Val Gly Tyr
85 90 95
Trp Ile Gln Ser Ile Arg Leu Phe Lys Arg Cys Arg Ser Trp Trp Ser
100 105 110
Phe Asn Pro Glu Ser Asn Ala Val Gly Ser Ile Leu Leu Thr Asn Gly
115 120 125
Gln Gln Cys Asn Phe Ala Ile Glu Ser Val Pro Met Val Leu Ser Pro
130 135 140
Ile Ile Lys Asn Gly Val Leu Tyr Cys Glu Gly Gln Trp Leu Ala Lys
145 150 155 160
Cys Glu Pro Asp His Leu Pro Lys Asp Ile Phe Val Cys Thr Pro Asp
165 170 175
Arg Arg Asn Ile Tyr Arg Met Val Gln Lys Tyr Thr Gly Asp Gln Ser
180 185 190
Gly Asn Lys Lys Arg Phe Ala Thr Phe Val Tyr Ala Lys Gln Ser Val
195 200 205
Asp Thr Gly Glu Leu Glu Ser Val Ala Thr Gly Gly Ser Ser Leu Tyr
210 215 220
Thr
225
<210> 84
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 84
ctttggatca ttaaacagac tttttaggtc tg 32
<210> 85
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 85
gtctgtttaa tgatccaaag tcccactag 29

Claims (15)

1. An H52IBV (infectious bronchitis virus) encoding a heterologous IBV S (spike) protein or fragment thereof.
2. An immunogenic composition comprising H52IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or fragment thereof.
3. An immunogenic composition comprising IBV (infectious bronchitis virus) according to claim 1.
4. The IBV or immunogenic composition of any one of claims 1 to 3 wherein the heterologous S protein or fragment thereof is from an IBV having a genotype or serotype selected from the group consisting of: arkansas (e.g., Arkansas 99), Brazil (e.g., BR-1, BR-2, 23/2013, IBV/Brasil/351/1984), California (e.g., California1734/04, California99), Connecticut, Delaware (e.g., Delaware 98), Dutch (e.g., D207, D212, D274, D3128, D3896, D8880, D1466), Florida, Georgia (e.g., Georgia GA-07, GA-08, GA-12, GA-13), Gray, Holte, Iowa (e.g., Iowa 97 and Iowa 69), Italy (e.g., Italy 02), JMK, IBT 5, Maine (e.g., Mannsylvania 209), Pennsylvania (e.g., Pennsylvania 1220/98, Pennsylvania/98), Wolk (e.g., Wolk 084/844), Qualv/843, CsJMK, IBT 3, Maine (e.g., CsJMvJV/8478), and CsJV/843), and CsJVg, JVg., CsJVJVg, JV/843, JVg, JVk, JV, CR 88).
5. The IBV or immunogenic composition of any one of claims 1 to 4 wherein the heterologous S protein or fragment thereof is from an IBV of a genotype or serotype selected from: 4/91, QX, Q1, Arkansas, variant 2 and Brazil.
6. The IBV or immunogenic composition of any one of claims 1 to 5 wherein the heterologous S protein or fragment thereof is selected from the group consisting of genotypes or serotypes of: 4/91, QX, Q1, Arkansas, variant 2 and Brazil, or said heterologous S protein or fragment thereof comprises or consists of the amino acid sequence: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.
7. The IBV or immunogenic composition of any one of claims 1-6, wherein the heterologous S protein is a full-length spike protein, or wherein a fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1077 amino acids in length.
8. The IBV or immunogenic composition of any one of claims 1 to 7 wherein the IBV is attenuated or the IBV is a recombinant IBV.
9. The immunogenic composition of any one of claims 2-8, wherein the immunogenic composition is a vaccine.
10. A kit comprising an IBV or immunogenic composition according to any one of claims 1 to 9.
11. A method of immunizing a subject comprising administering to the subject the immunogenic composition of any one of claims 2-9.
12.A method of treating or preventing clinical signs caused by IBV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition of any one of claims 2-9.
13. A method of reducing cilia retention in a subject in need thereof as compared to an unvaccinated control group of the same species, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition of any one of claims 2-9.
14. The method or use of any one of claims 11-13, wherein the subject is poultry.
15. The method or use of any one of claims 11-14, wherein the method results in an improvement in an efficacy parameter selected from the group consisting of: preventing or reducing cilia stasis, preventing or reducing rale, preventing or reducing egg drop, preventing or reducing kidney pathology, preventing or reducing watery diarrhea, preventing or reducing weight loss, reducing viral load, reducing virus shedding, or a combination thereof.
CN201980071601.2A 2018-10-31 2019-10-28 H52IBV vaccine with heterologous spike protein Pending CN112996536A (en)

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BR112021008390A2 (en) 2021-09-28
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